Method for detecting mutant dna

ABSTRACT

The present invention relates to a method for detecting of a mutant DNA using a probe, comprising: 
     (1) contacting a sample containing a single-stranded DNA which has a substituted nucleotide, a deleted nucleotide region, or an inserted nucleotide region (mutant-type DNA), or/and a wild-type single-stranded DNA (wild-type DNA) corresponding thereto with the probe which hybridizes with both single-stranded DNA, to form a hybrid with the mutant-type DNA (mutant-type hybrid) or/and a hybrid with a wild-type DNA (wild-type hybrid), wherein at least one of the obtained mutant-type hybrid and wild-type hybrid has the stem structure;
 
(2) separating the obtained mutant-type hybrid or/and wild-type hybrid by electrophoresis on the basis of presence or absence of the stem structure or difference in the stem structure; and
 
(3) detecting the presence or absence of the mutant-type DNA in the sample.

TECHNICAL FIELD

The present invention relates to a method for detecting of a mutant DNA(a mutant type DNA), wherein a hybrid of double-stranded DNA having acharacteristic shape is formed by hybridizing the single-stranded DNAwith a probe, and by separating the obtained hybrid by electrophoresisbased on said characteristic shape, the mutant-type DNA is detected.

BACKGROUND ART

In recent years, presence or absence of mutation in a particular genehas been emphasized in various fields. For example, in DNAidentification in forensics, determination of presence or absence of onenucleotide substitution by mutation or polymorphism in DNA is alreadywell known as an individual identification technique. In addition, alsoin the medical field, correlation between specific genetic polymorphismsand drug susceptibility has become known, and trials to reduce the riskof drug-induced health disaster by investigating specific geneticpolymorphisms have been done. Moreover, the detection of mutation in thegene is also employed for detection etc. of a mutant-type DNA holder ofa hereditary disorder and its importance is increasing rather thanbefore.

As for the conventional simple method for detecting single nucleotidesubstitution in DNA, SSCP method has been known. The SSCP method is amethod through the use of a fact that DNA fragment is amplified bypolymerase chain reaction (PCR) method, and the amplified product ismade to the single-stranded DNA by thermal melting, and in the coolingprocess after thermal melting, said single-stranded DNA forms base-pairspartially within a molecule; and in this method, the molecule havingsingle nucleotide mutation is changed to the shape of thesingle-stranded DNA, and based on the difference of mobility ofelectrophoresis from a wild-type DNA, separation and discrimination ofthe mutant-type DNA is performed. However, to maintain the molecularshape of single-strand DNA, this SSCP method needed to keep thetemperature constant during electrophoresis. Therefore, theelectrophoresis equipment is required to be provided with a circulationtype constant temperature system, and remained a problem that a big unitwould be needed.

And, as a method for solving the above-described problem on the SSCPmethod, a loop hybrid (LH) method in which single-stranded oligo DNA isadded to the reaction solution after the PCR reaction of DNA fragment tohybridize with DNA fragment, and the mutant-type DNA is discriminated byelectrophoresis on the basis of the structural difference of theobtained hybrid, has been proposed (JP-A-2007-61080).

-   Non-patent Literature 1: Orita, M. et al., Genomics 5, 874-879    (1989);-   Non-patent Literature 2: Matsukuma, S. et al., J. Mol. Diag. 8,    504-512 (2006);-   Patent Literature 1: JP-A-2007-61080.

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

The present inventors have found that in the above-described LH method,separation of the mutant-type DNA from the wild-type DNA byelectrophoresis was incomplete and sometimes the mutant-type DNA couldnot be detected, and have further investigated intensively to develop amethod which can separate the wild-type DNA and the mutant-type DNAcompletely and can detect the mutant-type DNA with high accuracy. As aresult, it was found that, by making the loop portion in the hybridobtained by LH method the stem structure, the hybrid derived from thewild-type DNA and the hybrid derived from the mutant-type DNA could beseparated easily by electrophoresis, and thus the present invention hasbeen completed. That is, the purpose of the present invention is toprovide a method for detecting the mutant-type DNA, in which the hybridof the wild-type DNA and the probe and the hybrid of the mutant-type DNAand the probe are formed, and these are separated by electrophoresisefficiently.

Means for Solving the Problems

That is, the present invention is a method for detecting the mutant-typeDNA using the probe, and relates to a method for detecting themutant-type DNA, comprising:

(1) contacting a sample containing a single-stranded DNA which has asubstituted nucleotide, a deleted nucleotide region, or an insertednucleotide region (mutant-type DNA), or/and a wild-type single-strandedDNA (wild-type DNA) corresponding thereto with the probe whichhybridizes with both single-stranded DNA, to form a hybrid with themutant-type DNA (mutant-type hybrid) or/and a hybrid with a wild-typeDNA (wild-type hybrid), wherein at least one of the obtained mutant-typehybrid and wild-type hybrid has the stem structure;(2) separating the obtained mutant-type hybrid or/and wild-type hybridby electrophoresis on the basis of presence or absence of the stemstructure or difference in the stem structure; and(3) detecting the presence or absence of the mutant-type DNA in thesample.

Effect of the Invention

According to the method of the present invention, with respect to theDNA in which mutant-type exists, it can be easily detected whether theDNA is the mutant-type DNA or not. In particular, even when it is themutant-type DNA which has been unable to perform separation anddetection by the conventional loop hybridization method, the presentinvention makes it possible to isolate and detect the hybrid of themutant-type DNA. Therefore, the methods of present invention can beapplied for various tests of clinical diagnosis, such as a test for thepresence or absence of cancer cells, a test for determination ofappropriateness of drug effectiveness by the presence or absence of aparticular gene, and the like. In addition, according to the presentinvention, even when it is the case where mutation occurs in codonsadjacent to each other (for example, codons 12 and 13) in KRAS genetictesting, it becomes possible to detect the both mutation with highaccuracy by a single measurement system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the result, obtained in Example 1, of electrophoresis carriedout by microchip electrophoresis method for the hybrids of the wild-typeDNA and the mutant-type DNA of KRAS gene which were prepared by the loophybrid method (LH method) using the probe which forms the hybrid havingthe stem structure.

FIG. 2 is the result, obtained in Comparative Example 1, ofelectrophoresis carried out by microchip electrophoresis method for thehybrids of the wild-type DNA and the mutant-type DNA of KRAS gene whichwere prepared by the LH method using the probe which forms the hybridnot having the stem structure.

FIG. 3 is the result, obtained in Example 2, of electrophoresis carriedout by polyacrylamide gel electrophoresis method for the hybrids of 7kinds of mutant-type DNA and wild-type DNA of KRAS gene which wereprepared by the LH method using the probe which forms the hybrid havingthe stem structure.

FIG. 4 is the result, obtained in Example 3, of electrophoresis, carriedout by polyacrylamide gel electrophoresis method for the hybrids of 12kinds of mutant-type DNA and wild-type DNA of KRAS gene which wereprepared by the LH method using the probe (IN-4) which forms the hybridhaving the stem structure.

FIG. 5 is the result, obtained in Example 4, of electrophoresis carriedout by microchip electrophoresis method for the hybrids of 12 kinds ofmutant-type DNA and wild-type DNA of KRAS gene which were prepared bythe LH method using the probe (IN-1) which forms the hybrid having thestem structure.

FIG. 6 shows the results, obtained in Example 5, of detection of thewild-type DNA (TA6) and mutant-type DNA (TA7) of UGT1A1 gene. Namely,the left panel shows the result of measurement of the hybrids preparedby the LH method for heterozygosity of the wild-type DNA (TA6) andmutant-type DNA (TA7); the middle panel shows the result of measurementof the hybrids prepared by the LH method for homozygosity of mutant-typeDNA (TA7); and the right panel shows the result of measurement of thehybrids prepared by the LH method for the homo of the wild-type DNA(TA6), respectively.

FIG. 7 shows the results, obtained in Example 6, of detection of thewild-type DNA (TA6) (lane 2) and mutant-type DNA (TA5) (lane 1), (TA7)(lane 3), and (TA8) (lane 4) of UGT1A1 gene. The left panel shows thosemeasured by SYBR Green I fluorescence detection, and the right panelshows those measured by detection of Cy5 fluorescence.

FIG. 8 is the figure, obtained in Example 7, which shows the result ofdetection of the wild-type DNA (TA6) and mutant-type DNA (TA7) of UGT1A1gene after the LH cycle of 0 to 4 times. The left figure shows theresult measured by fluorescence detection of Cy5; the right figure showsthe result measured by fluorescence detection of SYBR Green I.

FIG. 9 is the figure, obtained in Example 8, which shows the result ofdetection of SLH formation to the amplified region DNA havingfixed-length poly-T required to detect the poly-T polymorphisms in theTOMM40 gene, and its genotyping.

FIG. 10 illustrates stem and stem-loop structures in the hybridpertaining to the present invention.

FIG. 11 illustrates a nucleotide sequence of normal (wild-type) genomehaving a stem sequence which is capable of forming a stem structure andinserted between portions A and B of the genome in accordance with theinvention.

FIG. 12 is a table showing examples of combinations of a mutant-typehybrid and a wild-type hybrid according to the invention in the casewhere the mutant-type DNA has a substituted nucleotide, the probe hasthe nucleotide complementary to the normal nucleotide, and the hybridhas the stem structure in the probe side.

FIG. 13 is a table showing examples of combinations of a mutant-typehybrid and a wild-type hybrid according to the invention in the casewhere the mutant-type DNA has a substituted nucleotide, the probe hasthe nucleotide complementary to the substituted nucleotide, and thehybrid has the stem structure in the probe side.

FIG. 14 is a table showing examples of combinations of a mutant-typehybrid and a wild-type hybrid according to the invention in the casewhere the mutant-type DNA has a substituted nucleotide, the probe hasthe nucleotide which is complementary to the normal nucleotide, and atthe time when the probe has hybridized, a hybrid which has a stemsequence in the genome side is formed.

FIG. 15 is a table showing examples of combinations of a mutant-typehybrid and a wild-type hybrid according to the invention in the casewhere the mutant-type DNA has a substituted nucleotide, the probe hasthe nucleotide which is complementary to the substituted nucleotide, andat the time when these have hybridized, a hybrid which has a stemsequence in the genome side is formed.

FIGS. 16-27 illustrate various embodiments of stem structures inaccordance with the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The mutant-type DNA pertaining to the present invention is the one whichhas the nucleotide substitution, the deleted nucleotide region, or theinserted nucleotide region (hereinafter, these are sometimes referredcollectively and simply to as a mutated nucleotide region.), and anysingle-stranded DNA can be utilized if at least 100 to 1000 nucleotideof anteroposterior sequence of these mutated nucleotide region in thenucleotide sequence of its wild-type DNA correspond to the mutant-typeDNA is known. That is, genomic DNA fragment isolated from livingorganisms such as animal, microorganism, bacteria, and plant, DNAfragment which can be isolated from virus, and cDNA fragment synthesizedusing mRNA as a template are also included. Among these mutant-type DNA,oncogene derived from human cell is included as a preferable one. Inaddition, the chain length (chain length as a measuring target) ofmutant-type DNA which forms the hybrid is usually 50 to 2000 nucleotidespreferably 100 to 500 nucleotides. It should be noted that, theabove-described deleted nucleotide region represents the region in whichthe sequence of 1 to 200 nucleotides in the wild-type DNA iscontinuously missing. In addition, the above-described insertednucleotide region represents the region in which the sequence of 1 to200 nucleotides in the wild-type DNA is continuously inserted. It shouldbe noted that, since the probe which hybridizes with both mutant-typeDNA and wild-type DNA pertaining to the present invention may form thestem structure pertaining to the present invention by using a particularsubstituted nucleotide, deleted nucleotide region, or insertednucleotide region as a target, plural substituted nucleotide, deletednucleotide region, or inserted nucleotide region may exist other thanwhat is used as a target.

The wild-type DNA pertaining to the present invention is the DNA whichdoes not have mutation of the above-described mutant-type DNA pertainingto the present invention. The wild-type DNA pertaining to the presentinvention to be used in the methods of the present invention is the onein which the nucleotide sequence except for the mutant nucleotide regionis identical to the mutant-type DNA pertaining to the present invention,and as for its length, a comparable length with the mutant-type DNApertaining to the present invention is desirable to use, and usually itis 50 to 2000 nucleotides, preferably 100 to 500 nucleotides.

As for the above-described mutant-type DNA and the wild-type DNApertaining to the present invention, it is preferable that theabove-described DNA is purified as much as possible and unwantedsubstances except for nucleic acid fragment are removed. Specifically,for example, the one which is purified according to a routine methodsuch as Boom method employing the silica carrier (Boom et al., J. Clin.Microbiol. 28:495-503 (1990)), a method employing sodium iodide solution(Proc. Natl. Acad. Sci. USA 76-2, p615-619 (1979)) is preferable. Inaddition, the one which is the target DNA amplified by polymerase chainreaction (PCR reaction) well known per se, for example, by the methoddescribed in Nucleic Acids Research, 1991, Vol. 19, 3749, BioTechniques,1994, Vol. 16, 1134-1137, may be used.

The stem structure pertaining to the present invention is the one whichis composed of single-stranded nucleotide chain which does not hybridizewith the probe (does not form base pair) when it is formed in genomicDNA, and the one which is composed of single-stranded nucleotide chainwhich does not hybridize with the genomic DNA (does not form base pair)when it is formed in the probe, and which comprises a nucleotidesequence capable of forming base pair by itself. Said stem structure isthe one which is at least 2 or more consecutive base pairs, preferably 3or more consecutive base pairs, and it may be either consisted of thesequence capable of forming said base pair or comprising a sequence andloop sequence which are capable of forming aforementioned base pair. Itshould be noted that the loop sequence in the present invention is asingle-stranded nucleotide chain which cannot form base pair with theprobe, or a single-stranded nucleotide chain which cannot form base pairwith genomic DNA, and which cannot form base pair by itself in thehybridization of genomic DNA and the probe. In addition, as for theabove-described one which comprises a sequence capable of forming thebase pair and loop sequence, if it is the one which contains a sequencecapable of forming a base pair, the base pair may be located at any offront edge within the stem structure (most-distant position from thenucleotide sequence hybridizing with genomic DNA), the intermediateposition within the stem structure (the position surrounded with theloop sequence in the stem structure), or the end within the stemstructure (position nearest to the nucleotide sequence hybridizing withgenomic DNA). From the point of separation performance on theelectrophoresis of the mutant-type hybrid and the wild-type hybrid,among the above-described, the one which is consisted of only thesequence capable of forming base pair is preferable. It should be notedthat, in the hybrid pertaining to the present invention, deduced patterndiagrams of the stem structure is shown in FIG. 10. Sequentially fromthe left side, a pattern diagram of the stem structure which isconstituted only by a sequence capable of forming base pair; the stemstructure which is constituted by a sequence capable of forming basepair and a loop sequence, and the base pair is located at the end of thestem structure; the stem structure in which the base pair is located atfront edge of the stem structure; and the stem structure in which thebase pair is located at the intermediate position in the stem structure,are shown, respectively. A solid line represents the stem structure, adotted line represents a double-stranded nucleotide chain which genomicDNA and the probe form. In addition, a parallel part in the stemstructure represents a sequence capable of forming base pair and an arcrepresents the loop sequence.

The number of above-described base pairs which may be formed is usually2 to 20 bp, preferably 3 to 10 bp, and more preferably it is 3 to 5 bp.In addition, said base pair may comprise at least 2 consecutive basepairs, and may have multiple base pairs, and, what all are continued ispreferable. The above-described stem structure may be present either inthe probe side or in the genome side. If the stem structure is presentin the probe side, it is more desirable than present in a genome sidebecause the stem structure can be set up more freely by a probe design.The nucleotide chain length of the above-described stem structure isusually 4 to 60 mer, preferably 6 to 30 mer, and 6 to 20 mer is morepreferable.

It should be noted that, in the present invention, the stem structure inthe mutant-type hybrid and the stem structure in the wild-type hybridwill be different structure. That is, for example, if the mutant-typeDNA has a substituted nucleotide, and if the probe is designed to bindwith the substituted nucleotide but not to the normal nucleotide, and ifthe obtained hybrid has the stem structure in the probe side, itsmutant-type hybrid is presumed to form the same stem structure asdesigned in the probe side. On the other hand, in its wild-type hybrid,it is presumed that since the probe does not bind with the normalnucleotide, the stem structure in the hybrid will comprise a nucleotidenot binding with the normal nucleotide, and the stem structure in thewild-type hybrid will be longer by one nucleotide than the stemstructure (designed stem structure) in the mutant-type hybrid. Thus,when the stem structure is present in the probe side, in either one ofthe hybrid between the mutant-type hybrid and the wild-type hybrid, theprobe pertaining to the present invention provides the stem structuredifferent from that originally designed at the time of preparation ofthe probe, and in consequence, the mutant-type hybrid and the wild-typehybrid can be separated efficiently. It should be noted that, like anexample of the above-described wild-type hybrid, there may be a casewhere the stem structure in the hybrid is different from the designedone and a case where the stem structure does not form base pairs, namelythe case where the hybrid does not have the stem structure, Therefore,in the present invention, if only either the mutant-type hybrid or thewild-type hybrid has the stem structure, the mutant-type hybrid and thewild-type hybrid can be separated efficiently. In addition, for example,when a mutant-type DNA has the deleted nucleotide region, and thewild-type hybrid have the stem structure in the probe side, the stemstructure in the mutant-type hybrid will comprise an additionalnucleotide chain of the deleted nucleotide region to the stem structurein the wild-type hybrid. In addition, for example, when a mutant-typeDNA has the inserted nucleotide region, and the wild-type hybrid havethe stem structure in the genome side, the stem structure in themutant-type hybrid will comprise the additional inserted nucleotideregion to the stem structure in the wild-type hybrid. As describedabove, in the method of the present invention, the stem structure isformed in the different structures, and this make it possible toseparate the mutant-type hybrid from the wild-type hybrid efficiently byelectrophoresis on the basis of difference of the stem structure.

When the stem structure pertaining to the present invention is composedof a sequence capable of forming base pair and a loop sequence, it isconceived that there may be a case where a nucleotide in the loopsequence will bind with a complementary nucleotide in the base pair. Inthis case, it may become impossible to form base pairs, however, even insuch a case, according to the method of the present invention, since thestem structures of the mutant-type hybrid and the stem structure of thewild-type hybrid will be different structures, separation can beperformed efficiently. In addition, by the use of interaction (binding)between a nucleotide in the loop sequence and a nucleotide in the basepair, even when a mutant DNA with different type of substitutednucleotide is used, different stem structure can be formed, and as aconsequence, separation of mutant DNA with different type of substitutednucleotide is also possible.

The probe (hereinafter, sometimes written briefly as the probepertaining to the present invention) which hybridizes with bothmutant-type DNA and wild-type DNA pertaining to the present invention isthe one which hybridizes with the mutant-type DNA or the wild-type DNA,and forms respective hybrid, and on the occasion of hybridization, whichmakes at least one of the mutant-type hybrid and the wild-type hybridform the above-described stem structure, and when the stem structure isformed in both hybrids, it has been designed so that the stem structureof both hybrids will be different. Such probe pertaining to the presentinvention is usually 30 to 300 mer, preferably 50 to 150 mer. Inaddition, when the stem structure is formed in the probe side, the stemstructure can be set freely by designing of the probe, however, when thestem structure is formed in the genome side, the nucleotide sequencehaving the stem structure is retrieved from the nucleotide sequence ofgenome, the probe may be designed so that the stem structure is formedwith that portion. Although a specific example of the probe pertainingto the present invention will be described later together with aspecific example of the hybrid, for example, in the case where the DNAto be detected is a mutant-type DNA having a substituted nucleotide, andthe stem structure is intended to be formed in the probe side, the probemay be designed, for example, as the scheme described below. In the caseas shown in the scheme illustrated in FIG. 11, a single-strandednucleotide sequence of normal (wild-type) genome is divided into portionA and portion B with centering on the normal nucleotide corresponding tothe substituted nucleotide, and may be designed so that a sequence(corresponding sequence of the stem structure) which is capable offorming the stem structure is inserted in between portion A and portionB. It should be noted that M in the scheme represents a substitutednucleotide; N represents a normal nucleotide corresponding to thesubstituted nucleotide. In addition, portion A′ and portion B′ representnucleotide regions complementary to the portion A and portion B,respectively.

By designing in this way, for example, when the wild-type hybrid isformed, the stem structure as designed is formed. On the other hand, inthe mutant-type hybrid, since the substituted nucleotide and anucleotide in the probe which is complementary to the normal nucleotidedo not bind, it is conceived that the stem structure comprising onenucleotide complementary to the normal nucleotide is formed, and,consequently differs from wild-type stem structure. It should be notedthat, as described above, when the hybrid has the stem structure in theprobe side, as the sequence of the stem structure in the probepertaining to the present invention, a nucleotide sequence whichcomprises, for example, a palindrome (anagram) sequence, or a nucleotidesequence containing palindrome sequence, a nucleotide sequence in whichthe same number of nucleotides complementary to each other are connected(for example, AAAATTTT etc.), or a nucleotide sequence containing anucleotide sequence in which the same number of nucleotidescomplementary to each other are connected, are preferable.

The hybrid pertaining to the present invention is formed by hybridizingthe above-described probe pertaining to the present invention with thewild-type DNA or the mutant-type DNA, however, the wild-type hybrid andthe mutant-type hybrid are different in their stem structure asmentioned above. Namely, in the method of the present invention, becauseof forming both hybrids using the same probe, it is presumed that thenumber of coupled base pairs of the probe and DNA in the mutantnucleotide (region) of both hybrids differs; as a consequence, the stemstructures in both hybrids differs. Therefore, it becomes possible toseparate both hybrids easily by electrophoresis because this stemstructure is made different.

In addition, the hybrid pertaining to the present invention may furthercomprise the stem structure or the loop structure, among them preferablythe stem structure, in the probe side when the hybrid has the stemstructure in the opposite side of nucleotide chain having the stemstructure, namely, in the genomic DNA side, and in the DNA side when thehybrid has the stem structure in the probe side. By having the stemstructure or the loop structure in the opposite side of the nucleotidechain which has the stem structure, higher separation performance can beachieved. Such stem structure includes the same stem structurepertaining to the present invention as described above. In addition, theloop structure is composed of a single-stranded nucleotide chain whichcannot form the above-described stem structure and which does not formbase pair with the probe, or a single-stranded nucleotide chain whichdoes not form base pair with genomic DNA, and specifically, it is asingle-stranded nucleotide chain which does not form base pair by itselfor even if it forms base pair it is only one base pair, and does notform base pair with the probe, or a single-stranded nucleotide chainwhich does not form base pair with genomic DNA. The chain length thereofetc. may be the one according to the stem structure.

The above-described probe as well as the above-described hybrid may bedesigned appropriately and synthesized depending on the kind (thesingle-stranded DNA having the above-described substituted nucleotide,deleted nucleotide region, or inserted nucleotide region) of mutant-typeDNA to be a target of measurement, or on whether it hybridizes with themutant nucleotide region or a normal nucleotide or a normal nucleotideregion corresponding to the mutant nucleotide region, however, as forthe specific example of the probe, it will be described by dividing intocases as follows.

(I) In the case where DNA is the mutant-type DNA having a substitutednucleotide

-   -   (I-1) In the case where the hybrid which has the stem structure        in the probe side is formed        -   (a) In the case where the probe binds with a normal            nucleotide        -   (b) In the case where the probe binds with a substituted            nucleotide    -   (1-2) In the case where the hybrid which has the stem structure        in the genome side is formed        -   (a) In the case where the probe binds with a normal            nucleotide        -   (b) In the case where probe binds with a substituted            nucleotide            (II) In the case where DNA is the mutant-type DNA which has            the deleted nucleotide region    -   (II-1) In the case where the hybrid which has the stem structure        in the probe side is formed        -   (a) In the case where at least the mutant-type hybrid forms            the stem structure        -   (b) In the case where at least the wild-type hybrid forms            the stem structure    -   (II-2) In the case where the hybrid which has the stem structure        in the genome side is formed        (III) In the case where DNA is a mutant-type DNA which has the        inserted nucleotide region    -   (III-1) In the case where the hybrid which has the stem        structure in the genome side is formed        -   (a) In the case where at least the mutant-type hybrid forms            the stem structure        -   (b) In the case where at least the wild-type hybrid forms            the stem structure    -   (III-2) In the case where DNA has the stem structure in the        probe side        -   (a) In the case where at least the wild-type hybrid forms            the stem structure        -   (b) In the case where at least the mutant-type hybrid forms            the stem structure            (IV) In the case where the stem structure or the loop            structure is further formed in the chain opposite to the            nucleotide chain which has the stem structure

[Specific Example of the Probe and the Hybrid Pertaining to the PresentInvention]

(I) In the Case where DNA is a Mutant-Type DNA which has a SubstitutedNucleotide(I-1) In the Case where the Hybrid which has the Stem Structure in theProbe Side is Formed

In the case where a mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, the hybridwhich has the stem structure in the probe side is formed, for example,the probe pertaining to the present invention to be used has thefollowing structure.

3′ 1stSS₁-X′₁-St_(P1)-X′₂-X′₃-X′₄-X′₅-2ndSS₁ 5′, or3′ 1stSS₁-X′₁-X′₂-X′₃-X′₄-St_(P1)-X′₅-2ndSS₁ 5′[In said nucleotide sequences, 1stSS₁ and 2ndSS₁ represent thesingle-stranded nucleotide sequence which is complementary to thewild-type DNA and the mutant-type DNA, respectively; X′₁ and X′₅represent arbitrary nucleotide, respectively; and X′₂ to X′₄ represent anucleotide or a bond, and either one of them is the nucleotidecomplementary to the mutant nucleotide or the nucleotide complementaryto a normal nucleotide which corresponds to the mutant nucleotide; thenucleotide complementary to said mutant nucleotide or the nucleotidecomplementary to the normal nucleotide is located in the positioncorresponding to the substituted nucleotide in the mutant-type DNA orthe normal nucleotide in the wild-type DNA when hybridized. St_(P1)represents the sequence which forms the stem structure in the probe side(hereinafter, written briefly as the probe side stem sequence).].

The arbitrary nucleotide in the above-described X′₁ and X′₅ is anynucleotide consisted of adenine, guanine, thymine, or cytosine.Hereinafter, the arbitrary nucleotide in the present invention refers tothe same one.

The bond in X′₂ to X′₄ represents the case where the nucleotide is notrepresented but it combines the nucleotides on both adjacent sides. Forexample, when X′₂ is adenine (A), X′₃ is the bond and X′₄ is guanine(G), X′₂-X′₃-X′₄ represents A-G. Hereinafter, the bond in the presentinvention refers to the same one.

The probe side stem sequence pertaining to the present invention is theone which does not hybridize (does not form base pairs) with genomic DNAon the occasion of forming the hybrid, and the sequence in which thesingle-stranded nucleotide chain in the probe is capable of forming basepairs by itself, and is capable of forming the above-described stemstructure. Although said probe side stem sequence becomes the stemstructure by itself, it may form the stem structure by coupling with anucleotide or a nucleotide sequence adjacent to the stem sequence whichdoes not form base pair with genomic DNA. In this case, since theconstitution of the stem sequence is altered, there may be a situationthat the above-described base pairs cannot be formed, consequently, thestem structure pertaining to the present invention cannot be constituted(but forms the loop structure), Therefore, it is necessary to design sothat at least any one of the mutant-type hybrid and the wild-type hybridhas the stem structure pertaining to the present invention.

The probe side stem sequence pertaining to the present invention is theone which usually consists of 4 to 60 mer, preferably 6 to 20 mer. Inaddition, the base pair in the stem structure at the time when saidprobe side stem sequence forms the stem structure includes the one whichconsists of a consecutive double-stranded base pair of usually 2 to 20bp, preferably 3 to 10 bp and more preferably 3 to 5 bp, or the onewhich comprises a consecutive double-stranded base pair of usually 2 to20 bp, preferably 3 to 10 bp and more preferably 3 to 5 bp and a loopsequence. Among them, the one which consists of a consecutivedouble-stranded base pair is preferable.

When the above-described probe is hybridized with mutant-type DNA, thefollowing mutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₁-X′₁-St_(P1)-X′₂-X′₃-X′₄-X′₅-2ndSS₁ 5′Genome: 5′ 1stDS₁-X₁-X₂-X₃-X₄-X₅-2ndDS₁ 3′orProbe: 3′ 1stSS₁-X′₁-X′₂-X′₃-X′₄-St_(P1)-X′₅-2ndSS₁ 5′Genome: 5′ 1stDS₁-X₁-X₂-X₃-X₄-X₅-2ndDS₁ 3′(In said hybrid, 1stSS₁ and 2ndSS₁ are the same as described above, and1stDS₁ and 2ndDS₁ represent nucleotide sequence region which formscomplementary double strand with 1stSS₁ and 2ndSS₁, respectively;although X′₁ to X′₅ are the same as described above, X₁ and X₅ representnucleotide, X′₁ and X₁, and X′₅ and X₅ are complementary nucleotides,respectively, and X₂ to X₄ represent the nucleotide or the bond, and anyone of them is the substituted nucleotide, and it is the nucleotide orthe bond complementary to the corresponding X′₂ to X′₄. In this regard,however, when any of X′₂ to X′₄ in the probe is the nucleotidecomplementary to the normal nucleotide, it will not be complementary tothe substituted nucleotide of any of corresponding X₂ to X₄.).

In addition, when the above-described probe is hybridized with thewild-type DNA, the following mutant-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₁-X′₁-St_(P1)-X′₂-X′₃-X′₄-X′₅-2ndSS₁ 5′Genome: 5′ 1stDS₁-X₁-Y₂-Y₃-Y₄-X₅-2ndDS₁ 3′orProbe: 3′ 1stSS₁-X′₁-X′₂-X′₃-X′₄-St_(P1)-X′₅-2ndSS₁ 5′Genome: 5′ 1stDS₁-X₁-Y₂-Y₃-Y₄-X₅-2ndDS₁ 3′(In said hybrid, although 1stSS₁, 2ndSS₁ 1stDS₁, 2ndDS₁, X′₁ to X′₅, andX₁ and X₅ are the same as described above, X′₁ and X₁, and X′₅ and X₅are complementary nucleotides, respectively; Y₂ to Y₄ represent thenucleotide or the bond, and any one of them is a normal nucleotidecorresponding to mutant nucleotide, and it is the nucleotide or the bondcomplementary to corresponding X′₂ to X′₄. In this regard, however, whenany one of X′₂ to X′₄ in the probe is the nucleotide complementary to asubstituted nucleotide, it will not be complementary to the normalnucleotide of any one of corresponding Y₂ to Y₄.).

The case where the mutant-type DNA has a substituted nucleotide and hasthe stem structure in the probe side will be explained as follows byfurther dividing the case into (a) a case where the probe has thenucleotide complementary to a normal nucleotide and (b) a case where theprobe has the nucleotide complementary to a substituted nucleotide.

(I-1) (a) in the Case where the Hybrid which has the Stem Structure inthe Probe Side is Formed, and the Probe Binds with a Normal Nucleotide

In the case where the mutant-type DNA is a DNA which has a substitutednucleotide, and at the time when the probe has hybridized, the probebinds with normal nucleotide and forms the hybrid which has the stemstructure in the probe side, said probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₁₁-X′₁₁-St_(P11)-X′₁₂-X′₁₃-X′₁₄-X′₁₅-2ndSS₁₁ 5′or3′ 1stSS₁₁-X′₁₁-X′₁₂-X′₁₃-X′₁₄-St_(P11)-X′₁₅-2ndSS₁₁ 5′(In said nucleotide sequence, 1stSS₁₁ and 2ndSS₁₁ represent asingle-stranded nucleotide sequence which is complementary to thewild-type DNA and the mutant-type DNA, respectively; X′₁₁ and X′₁₅represent each arbitrary nucleotide; and X′₁₂ to X′₁₄ represent thenucleotide or the bond, and either one of them is the nucleotidecomplementary to the normal nucleotide which corresponds to the mutantnucleotide, and at the time when it has hybridized, said nucleotidecomplementary to the normal nucleotide is located in the positioncorresponding to the substituted nucleotide of the mutant-type DNA orthe normal nucleotide of the wild-type DNA. St_(P11) represents theprobe side stem sequence.).

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₁₁-X′₁₁-X′₁₂-X′₁₃-X′₁₄-St_(P11)-X′₁₅-2ndSS₁₁ 5′Genome: 5′ 1stDS₁₁-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-2ndDS₁₁ 3′orProbe: 3′ 1stSS₁₁-X′₁₁-St_(P11)-X′₁₂-X′₁₃-X′₁₄-X′₁₅-2ndSS₁₁ 5′Genome: 5′ 1stDS₁₁-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-2ndDS₁₁ 3′(In said hybrid, X′₁₁ to X′₁₅, St_(P11) are the same as described above;1stSS₁₁, 2ndSS₁₁ are the same as described above, and 1stDS₁₁ and2ndDS₁₁ represent the nucleotide sequence region which formscomplementary double strand with 1stSS₁₁ and 2ndSS₁₁, respectively; X₁₁and X₁₅ represent nucleotide; X′₁₁ and X₁₁, and X′₁₅ and X₁₅ arecomplementary nucleotides; X₁₂ to X₁₄ represent the nucleotide or thebond, and either one of them is the substituted nucleotide; thesubstituted nucleotide in the X₁₂ to X₁₄ cannot be the nucleotidecomplementary to the corresponding X′₁₂ to X′₁₄, however, the rest arethe nucleotide or the bond complementary to the corresponding X′₁₂ toX′₁₄.).

It should be noted that, since any one of X′₁₂ to X′₁₄ in theabove-described mutant-type hybrid is the nucleotide complementary tothe normal nucleotide, it does not form a base pair with substitutednucleotide in the X₁₂ to X₁₄. Therefore, when the nucleotide is presentin between the probe side stem sequence and the nucleotide complementaryto the normal nucleotide, even if the nucleotide is the nucleotidecomplementary to the genome side nucleotide, its binding will beunstable, and hence unable to form the base pair. In consequence, insome instances, X′₁₂ to X′₁₄ also become unable to form base pairs, andsuch nucleotide which is unable to form base pair is coupled to the stemsequence. It should be noted that, in this case, these nucleotides mayform the stem structure depending on the combination of nucleotideswhich have become unable to form stem sequence and base pair, orotherwise may form a simple loop structure. For example, as a schemedescribed below, when X₁₃ is substituted nucleotide (in the scheme,represented by M), and X′₁₃ is the nucleotide (in the scheme,represented by N′) complementary to normal nucleotide, in normalcircumstances, X₁₂ and X′₁₂ form a base pair, however, since nucleotidesin adjacent both sides do not form base pairs with nucleotides in genomeside, the entire St_(P11)-X′₁₂-N′ (X′₁₃) does not form base pair withthe genome side of mutant-type DNA, and eventually the stem structure isformed by these.

In addition, if the above-described probe is hybridized with thewild-type DNA, the following wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₁₁-X′₁₁-St_(P11)-X′₁₂-X′₁₃-X′₁₄-X′₁₅-2ndSS₁₁ 5′Genome: 5′ 1stDS₁₁-X₁₁-Y₁₂-Y₁₃-Y₁₄-X₁₅-2ndSS₁₁ 3′orProbe: 3′ 1stSS₁₁-X′₁₁-X′₁₂-X′₁₃-X′₁₄-St_(P1)-X′₁₅-2ndSS₁₁ 5′Genome: 5′ 1stDS₁₁-X₁₁-Y₁₂-Y₁₃-Y₁₄-X₁₅-2ndDS₁₁ 3′(In said hybrid, 1stSS₁₁, 2ndSS₁₁ are same as described above, and1stDS₁₁, 2ndDS₁₁, X′₁₁ to X′₁₅, X₁₁, X₁₅ and St_(P11) are also the sameas described above; however, X₁₁ and X₁₅ are the nucleotidescomplementary to X′₁₁ and X′₁₅, respectively; and Y₁₂ to Y₁₄ representthe nucleotide or the bond, and any one of them is the normal nucleotidecorresponding to the mutant nucleotide, and it is the nucleotide or thebond complementary to the corresponding to X′₁₂ to X′₁₄.).

In the above-described wild-type hybrid, the stem structure is formedonly by a stem sequence which is designed in the probe. In the methodfor detecting mutant-type DNA of the present invention, since saidwild-type hybrid will have at least the stem structure, theabove-described mutant-type hybrid may not have stem structure. Itshould be noted that, even when the above-described mutant-type hybridhas the stem structure, it will have the stem structure different fromsaid wild-type hybrid.

In the case where the above-described mutant-type DNA is the one whichhas a substituted nucleotide, and the probe has the nucleotidecomplementary to the normal nucleotide, and the hybrid having the stemstructure in the probe side is formed, the specific example ofcombination of the mutant-type hybrid and the wild-type hybrid includes,for example, the combinations as listed in the table shown in FIG. 12.

In the table shown in FIG. 12, 1stSS and 2ndSS represent thesingle-stranded nucleotide sequence which is complementary to thewild-type DNA and the mutant-type DNA, respectively; 1stDS and 2ndDSrepresent the nucleotide sequence regions which form complementarydouble strand with 1stSS and 2ndSS, respectively; X represents thenucleotide which becomes complementary between the probe and the genomicDNA; M represents the substituted nucleotide, N represents the normalnucleotide corresponding to the substituted nucleotide, and N′represents the nucleotide complementary to the normal nucleotide, and Strepresents the probe side stem sequence. In addition, the area enclosedwith square (□) represents that it does not form base pair with genomicDNA, but forms the stem structure or the loop structure.

(I-1) (b) in the Case where the Hybrid which has the Stem Structure inthe Probe Side is Formed, and the Probe Binds with a SubstitutedNucleotide

In the case where the mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, the probebinds with substituted nucleotide and forms the hybrid which has thestem structure in the probe side, said probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₂₁-X′₂₁-St_(P21)-X₂₂-X′₂₃-X′₂₄-X′₂₅-2ndSS₂₁ 5′or3′ 1stSS₂₁-X′₂₁-X′₂₂-X′₂₃-X′₂₄-St_(P21)-X′₂₅-2ndSS₂₁ 5′(In said nucleotide sequence, 1stSS₂₁ and 2ndSS₂₁ represent thesingle-stranded nucleotide sequence which is complementary to thewild-type DNA and the mutant-type DNA, respectively; X′₂₁ and X′₂₅represent arbitrary nucleotide; and X′₂₂ to X′₂₄ represent thenucleotide or the bond, and any one of them is the nucleotidecomplementary to the substituted nucleotide, and at the time when it hashybridized, said nucleotide complementary to the substituted nucleotideis located in the position corresponding to the substituted nucleotideof the mutant-type DNA or the normal nucleotide of the wild-type DNA.St_(P21) represents the probe side stem sequence.).

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₂₁-X′₂₁-St_(P21)-X′₂₂-X′₂₃-X′₂₄-X′₂₅-2ndSS₂₁ 5′Genome: 5′ 1stDS₂₁-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-2ndSS₂₁ 3′orProbe: 3′ 1stSS₂₁-X′₂₁-X′₂₂-X′₂₃-X′₂₄-St_(P21)-X′₂₅-2ndSS₂₁ 5′Genome: 5′ 1stDS₂₁-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-2ndDS₂₁ 3′(In said hybrid, 1stSS₂₁, 2ndSS₂₁, X′₂₁ to X′₂₅, and St_(p21) are thesame as described above, and 1stDS₂₁ and 2ndDS₂₁ represent thenucleotide sequence region which forms complementary double strand with1stSS₂₁, 2ndSS₂₁, respectively; X₂₁ and X₂₅ represent nucleotide; X′₂₁and X₂₁, and X′₂₅ and X₂₅ represent complementary nucleotides; and X₂₂to X₂₄ represent the nucleotide or the bond, and any one of them is thesubstituted nucleotide, and it is the nucleotide or the bondcomplementary to the corresponding to X′₂₂ to X′₂₄.).

In said mutant-type hybrid, the stem structure is formed only by thestem sequence which is designed by the probe. In the method fordetecting mutant-type DNA of the present invention, since saidmutant-type hybrid will have at least the stem structure, the wild-typehybrid described below may not have stem structure. It should be notedthat, even when the wild-type hybrid described below has the stemstructure; it will have the stem structure different from saidmutant-type hybrid.

In addition, if the above-described probe is hybridized with thewild-type DNA, the following wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₂₁-X′₂₁-St_(P21)-X′₂₂-X′₂₃-X′₂₄-X′₂₅-2ndSS₂₁ 5′Genome: 5′ 1stDS₂₁-X₂₁-Y₂₂-Y₂₃-Y₂₄-X₂₅-2ndSS₂₁ 3′orProbe: 3′ 1stSS₂₁-X′₂₁-X′₂₂-X′₂₃-X′₂₄-St_(P21)-X′₂₅-2ndSS₂₁ 5′Genome: 5′ 1stDS₂₁-X₂₁-Y₂₂-Y₂₃-Y₂₄-X₂₅-2ndDS₂₁ 3′(In said hybrid, 1stSS₂₁, 2ndSS₂₁, 1stDS₂₁, 2ndDS₂₁, X′₂₁ to X′₂₅, X₂₁,X₂₅ and St_(P21) are the same as described above; however X₂₁ and X₂₅are the nucleotides complementary to X′₂₁ and X′₂₅, respectively; Y₂₂ toY₂₄ represent the nucleotide or the bond, and any one of them is thenormal nucleotide corresponding to a mutant nucleotide, and the rest ofY₂₂ to Y₂₄ is the bond or the nucleotide complementary to thecorresponding to Y′₂₂ to Y′₂₄.).

It should be noted that, since any one of X′₂₂ to X′₂₄ in theabove-described wild-type hybrid is the nucleotide complementary to thesubstituted nucleotide, it does not form the base pair with any one ofnormal nucleotide in the Y₂₂ to Y₂₄. In consequence, in some instances,X′₂₂ to X′₂₄ are unable to form the base pair due to the effect ofstructure of the stem sequence, and such nucleotide which is unable toform the base pair is coupled to the probe side stem sequence. It shouldbe noted that, in this case, these nucleotides may form the stemstructure depending on the combination of nucleotides which have becomeunable to form stem sequence and base pair, or otherwise may form theloop structure simply.

In the case where the above-described mutant-type DNA is the one whichhas the substituted nucleotide, and the probe has the nucleotidecomplementary to the substituted nucleotide, and it forms the hybridhaving the stem sequence in the probe side, the specific example ofcombination of the mutant-type hybrid and the wild-type hybrid includes,for example, the combinations as listed in the table shown in FIG. 13.

In the table shown in FIG. 13, 1stSS and 2ndSS represent thesingle-stranded nucleotide sequence which is complementary to thewild-type DNA and the mutant-type DNA, respectively; 1stDS and 2ndDSrepresent the nucleotide sequence regions which form complementarydouble strand with 1stSS and 2ndSS, respectively; X represents thenucleotide which becomes complementary each other between the probe andgenomic DNA; M represents a substituted nucleotide, N represents anormal nucleotide corresponding to the substituted nucleotide, and M′represents the nucleotide complementary to a substituted nucleotide, andSt represents the probe side stem sequence. In addition, the areaenclosed with square (□) represents that it does not form base pair withgenomic DNA, but forms the stem structure or the loop structure.

As mentioned above, in the case where the mutant-type DNA is the DNAwhich has a substituted nucleotide, and the hybrid which has the stemstructure in the probe side is formed, the hybrid further comprises theone which forms a further the stem structure or the loop structure inthe genome side. The details will be described in (IV).

(1-2) In the Case where the Hybrid which has the Stem Structure in theGenome Side is Formed

In the case where a mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, it forms thehybrid which has the stem structure in the genome side, the probepertaining to the present invention to be used has, for example, thefollowing structure.

3′ 1stSS₃₁-X′₃₁-X′₃₂-X′₃₃-2ndSS₃₁ 5′(1stSS₃₁ and 2ndSS₃₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₃₁, X′₃₂, and X′₃₃ represent arbitrary nucleotiderespectively; and X′₃₂ represent the nucleotide which is complementaryto the substituted nucleotide or the nucleotide complementary to thenormal nucleotide.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₃₁-X′₃₁-X′₃₂-X′₃₃-2ndSS₃₁ 5′Genome: 5′ 1stDS₃₁-X₃₁-M₃₂-St_(G31)-X₃₃-2ndDS₃₁ 3′orProbe: 3′ 1stSS₃₁-X′₃₁-X′₃₂-X′₃₃-2ndSS₃₁ 5′Genome: 5′ 1stDS₃₁-X₃₁-St_(G31)-M₃₁-X₃₃-2ndDS₃₁ 3′(In said hybrid, 1stSS₃₁, 2ndSS₃₁, X′₃₁ to X′₃₃ are the same asdescribed above. 1stDS₃₁, 2ndDS₃₁ represent the nucleotide sequenceregion which forms complementary double strand together with 1stSS₃₁,2ndSS₃₁, respectively; X₃₁ and X₃₃ each represent nucleotide, which arenucleotides complementary to X′₃₁ and X′₃₃, respectively; M₃₁ representssubstituted nucleotide; St_(G31) represents a sequence (hereinafter,sometimes abbreviated as genome side stem sequence) which forms the stemstructure in the genome side.

In said hybrid, when X′₃₂ is the nucleotide which is complementary tothe substituted nucleotide, the genome side stem sequence forms the stemstructure; and when X′₃₂ is the nucleotide which is complementary to thenormal nucleotide, the genome side stem sequence and the substitutednucleotide are coupled to form the stem structure or the loop structure.

Similarly as the case of the above-described probe side stem sequence,the above-described genome side stem sequence is the sequence which doesnot hybridize (does not form base pair) with the probe at the time whenthe hybrid is formed, and the sequence which the single-strandednucleotide chain in the genome forms the base pairs by itself, and theone which forms the above-described stem structure. Although said stemsequence becomes the stem structure by itself, when X′₃₂ is thenucleotide complementary to the normal nucleotide, the stem structure orthe loop structure is formed with St_(G31)-M₃₁. It should be noted that,when the loop structure is formed, it is necessary to design so that thewild-type hybrid has the stem structure pertaining to the presentinvention.

The genome side stem sequence usually consists of 6 to 60 mer,preferably 6 to 10 mer, and the base pair in the stem structure at thetime when said genome side stem sequence forms the stem structureincludes the one which consists of a consecutive double-stranded basepairs of usually 2 to 20 bp, preferably 3 to 5 bp, or the loop structurewhich has the consecutive double-stranded base pairs of usually 3 to 20bp, preferably 3 to 5 bp. Among them, the one which consists of aconsecutive double-stranded base pairs of 3 to 20 bp, preferably 3 to 5bp is preferable.

When the above-described probe is hybridized with the wild-type DNA, thefollowing mutant-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₃₁-X′₃₁-X′₃₂-X′₃₃-2ndSS₃₁ 5′Genome: 5′ 1stDS₃₁-X₃₁-St_(G31)-N₃₁-X₃₃-2ndDS₃₁ 3′orProbe: 3′ 1stSS₃₁-X′₃₁-X′₃₂-X′₃₃-2ndSS₃₁ 5′Genome: 5′ 1stDS₃₁-X₃₁-N₃₁-St_(G31)-X₃₃-2ndDS₃₁ 3′(1stSS₃₁, 2ndSS₃₁, 1stDS₃₁, 2ndDS₃₁, X′₃₁ to X′₃₃, X₃₁, X₃₃, andSt_(G31) are the same as described above. N₃₁ represents the normalnucleotide corresponding to the substituted nucleotide.).

In said hybrid, when X′₃₂ is the nucleotide complementary to the normalnucleotide, the genome side stem sequence forms the stem structure; andwhen X′₃₂ is the nucleotide complementary to the substituted nucleotide,the genome side stem sequence and the substituted nucleotide are coupledto form the stem structure or the loop structure.

(I-2) (a) in the Case where the Hybrid which has the Stem Structure inthe Genome Side is Formed, and the Probe Binds with a Normal Nucleotide

In the case where the mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, the probebinds with a normal nucleotide and form the hybrid which has the stemstructure in the genome side, the probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₄₁-X′₄₁-N′₄₁-X′₄₃-2ndSS₄₁ 5′(1stSS₄₁, 2ndSS₄₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₄₁ and X′₄₃ represent the nucleotides, respectively;N′₄₁ represents the nucleotide complementary to the normal nucleotide.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₄₁-X′₄₁-N′₄₁-X′₄₃-2ndSS₄₁ 5′Genome: 5′ 1stDS₄₁-X₄₁-M₄₁-St_(G41)-X₄₃-2ndSS₄₁ 3′orProbe: 3′ 1stSS₄₁-X′₄₁-N′₄₁-X′₄₃-2ndSS₄₁ 5′Genome: 5′ 1stDS₄₁-X₄₁-St_(G41)-M₄₁-X₄₃-2ndSS₄₁ 3′(In said hybrid, 1stSS₄₁, 2ndSS₄₁, X′₄₁, X′₄₃, and N′₄₁ are the same asdescribed above. 1stDS₄₁ and 2ndDS₄₁ represent the nucleotide sequenceregion which forms complementary double strand with 1stSS₄₁ and 2ndSS₄₁,respectively; X₄₁ and X₄₃ represent nucleotide, and these are thenucleotides complementary to X′₄₁ and X′₄₃, respectively; M₄₁ representsthe substituted nucleotide; and St_(G41) represents the genome side stemsequence.)

In the above-described hybrid, since the substituted nucleotide M₄₁cannot form base pairs with N′₄₁, the stem structure or the loopstructure is formed by coupling with St_(G41).

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₄₁-X′₄₁-N′₄₁-X′₄₃-2ndSS₄₁ 5′Genome: 5′ 1stDS₄₁-X₄₁-St_(G41)-N₄₁-X₄₃-2ndDS₄₁ 3′orProbe: 3′ 1stSS₄₁-X′₄₁-X′₄₂-X′₄₃-2ndSS₄₁ 5′Genome: 5′ 1stDS₄₁-X₄₁-N₄₁-St_(G41)-X₄₃-2ndDS₃₁ 3′(1stSS₄₁, 2ndSS₄₁, 1stDS₄₁, 2ndDS₄₁, X′₄₁, X′₄₂ and X′₄₃, X₄₁, X₄₃, andSt_(G41) are the same as described above. N₄₁ represents the normalnucleotide corresponding to the substituted nucleotide.)

In said hybrid, the genome side stem sequence forms the stem structure.

In the case where the mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, the hybridwhich has a stem sequence in the genome side is formed; the specificexample of combination of the mutant-type hybrid and the wild-typehybrid includes, for example, the combinations as listed in the tableshown in FIG. 14.

In the table shown in FIG. 14, 1stSS, 2ndSS represent thesingle-stranded nucleotide sequence which is complementary to thewild-type DNA and mutant-type DNA, respectively; 1stDS, 2ndDS representthe nucleotide sequence regions which form complementary double strandwith 1stSS, 2ndSS, respectively; X represents the nucleotide whichbecomes complementary between the probe and genomic DNA; M representsthe substituted nucleotide, N represents the normal nucleotidecorresponding to the substituted nucleotide, and N′ represents thenucleotide complementary to the normal nucleotide, and (St) representsthe genome side stem sequence. In addition, the area enclosed withsquare (□) represents that it does not form base pair with the probe,but forms the stem structure or the loop structure.

(I-2) (b) in the Case where the Hybrid which has the Stem Structure inthe Genome Side is Formed, and the Probe Binds with a SubstitutedNucleotide

In the case where the mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, the probebinds with substituted nucleotide and forms the hybrid which has thestem structure in the genome side, the probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₅₁-X′₅₁-M′₅₁-X′₅₃-2ndSS₅₁ 5′(1stSS₅₁, 2ndSS₅₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₅₁ and X′₅₃ represent the nucleotides, respectively;M′₅₁ represents the nucleotide complementary to the substitutednucleotide.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₅₁-X′₅₁-M′₅₁-X′₅₃-2ndSS₅₁ 5′Genome: 5′ 1stDS₅₁-X₅₁-M₅₁-St_(G51)-X₅₃-2ndDS₅₁ 3′orProbe: 3′ 1stSS₅₁-X′₅₁-M′₅₁-X′₅₃-2ndSS₅₁ 5′Genome: 5′ 1stDS₅₁-X₅₁-St_(G51)-M₅₁-X₅₃-2ndDS₅₁ 3′(In said hybrid, 1stSS₅₁, 2ndSS₅₁, X′₅₁, X′₅₃, and M′₅₁ are the same asdescribed above. 1stDS₅₁, 2ndDS₅₁ represent the nucleotide sequenceregions which form base pairs with 1stSS₅₁ and 2ndSS₅₁, respectively;X₅₁ and X₅₃ represent nucleotide, and X′₅₁ and X₅₁, and X′₅₃ and X₅₃ arecomplementary nucleotides, respectively; M₅₁ represents the substitutednucleotide; and St_(G41) represents the genome side stem sequence.)

In said hybrid, the genome side stem sequence forms the stem structure.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₅₁-X′₅₁-M′₅₁-X′₅₃-2ndSS₅₁ 5′Genome: 5′ 1stDS₅₁-X₅₁-St_(G51)-N₅₁-X₅₃-2ndDS₅₁ 3′orProbe: 3′ 1stSS₅₁-X′₅₁-M′₅₁-X′₅₃-2ndSS₅₁ 5′Genome: 5′ 1stDS₅₁-X₅₁-N₅₁-St_(G51)-X₅₃-2ndDS₅₁ 3′(In said hybrid, 1stSS₅₁, 2ndSS₅₁, 1stDS₅₁, 2ndDS₅₁, X′₅₁ to X′₅₃ arethe same as described above. X₅₁ and X₅₃ represent the nucleotides,respectively, and these are nucleotides complementary to X′₅₁ and X′₅₃,respectively; N₅₁ represents the normal nucleotide corresponding to thesubstituted nucleotide; and St_(G51) represents the genome side stemsequence.)

In the above-described hybrid, since N₅₁ cannot form base pairs withM′₅₁, the stem structure or the loop structure is formed by couplingwith St_(G51).

In the case where the mutant-type DNA is the one which has a substitutednucleotide, and the probe has the nucleotide which is complementary tothe substituted nucleotide, and at the time when these have hybridized,the hybrid which has a stem sequence in the genome side is formed; thespecific example of combination of the mutant-type hybrid and thewild-type hybrid includes, for example, the combinations as listed inthe table shown in FIG. 15.

In the table shown in FIG. 15, 1stSS, 2ndSS represent thesingle-stranded nucleotide sequence which is complementary to thewild-type DNA and mutant-type DNA, respectively; 1stDS, 2ndDS representthe nucleotide sequence regions which form complementary double strandwith 1stSS, 2ndSS, respectively; X represents the nucleotide which iscomplementary each other between the probe and genomic DNA; M representsa substituted nucleotide, N represents a normal nucleotide correspondingto the substituted nucleotide, and M′ represents the nucleotidecomplementary to the substituted nucleotide, and (St) represents thegenome side stem sequence. In addition, the area enclosed with square(□) represents that it does not form base pair with the probe, but formsthe stem structure or the loop structure.

As mentioned above, in the case where the mutant-type DNA is a DNA whichhas a substituted nucleotide, and the hybrid which has the stemstructure in the probe side is formed, the hybrid further comprises thehybrid which forms further the stem structure or the loop structure inthe genome side. The details will be described in (IV).

(II) In the Case where DNA is a Mutant-Type DNA which has the DeletedNucleotide Region(II-1) in the Case where the Hybrid which has the Stem Structure in theProbe Side is Formed

(a) In the Case where at Least the Mutant-Type Hybrid Forms the StemStructure

In the case where the mutant-type DNA is the one which has the deletednucleotide region, and at the time when the probe has hybridized, thestem structure is formed in the probe side, and at least the mutant-typehybrid forms the stem structure; the probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₂₁₁-X′₂₁₁-DM′-X′₂₁₂-2ndSS₂₁₁ 5′(1stSS₂₁₁, 2ndSS₂₁₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₂₁₁ and X′₂₁₂ represent arbitrary nucleotides,respectively; DM′ represents the nucleotide sequence which iscomplementary to the deleted nucleotide region, and which forms the stemstructure.)

The above-described deleted nucleotide region means the nucleotideregion in the wild-type DNA which is deleted in the mutant-type DNA.

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₂₁₁-X′₂₁₁-DM′-X′₂₁₂-2ndSS₂₁₁ 5′Genome: 5′ 1stDS₂₁₁-X₂₁₁-X₂₁₂-2ndDS₂₁₁ 3′(In said hybrid, 1stSS₂₁₁, 2ndSS₂₁₁, X′₂₁₁, X′₂₁₂, and DM′ are the sameas described above. 1stDS₂₁₁, 2ndDS₂₁₁ represent the nucleotide sequenceregion which forms complementary double strand with 1stSS₂₁₁, 2ndSS₂₁₁,respectively; X₂₁₁ and X₂₁₂ represent respective nucleotides, and X′₂₁₁and X₂₁₁, and X′₂₁₂ and X₂₁₂ are complementary nucleotides,respectively.)

In the above-described hybrid, the nucleotide sequence complementary tothe deleted nucleotide region forms the stem structure.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₂₁₁-X′₂₁₁-DM′-X′₂₁₂-2ndSS₂₁₁ 5′Genome: 5′ 1stDS₂₁₁-X₂₁₁-DM-X₂₁₂-2ndDS₂₁₁ 3′(In said hybrid, 1stSS₂₁₁, 2ndSS₂₁₁, 1stDS₂₁₁, 2ndDS₂₁₁, X′₂₁₁ and X′₂₁₂are the same as described above. X₂₁₁ and X₂₁₂ represent nucleotide,respectively, and these are complementary to X′₂₁₁ and X′₂₁₂,respectively. DM represents the deleted nucleotide region; and DM′ isthe nucleotide complementary to the deleted nucleotide region, whichforms base pair with DM).

The above-described hybrid does not form the stem structure because theprobe and genome can form base pairs completely.

(b) In the Case where at Least the Wild-Type Hybrid Forms the StemStructure

In the case where the mutant-type DNA is a DNA which has the deletednucleotide region, and at the time when the probe is hybridized, thestem structure is formed in the probe side, and at least the wild-typehybrid forms the stem structure (the wild-type hybrid forms a designedstem structure); the probe pertaining to the present invention to beused has, for example, the following structure.

3′ 1stSS₂₂₁-X′₂₂₁-DM′-St_(P221)-X′₂₂₂-2ndSS₂₂₁ 5′(1stSS₂₂₁, 2ndSS₂₂₁ represents the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; and X′₂₂₁ and X′₂₂₂ represent the nucleotides,respectively. DM′ represents the nucleotide sequence which iscomplementary to the deleted nucleotide region and St_(p221) representsthe probe side stem sequence.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₂₂₁-X′₂₂₁-DM′-St_(P221)-X′₂₂₂-2ndSS₂₂₁ 5′Genome: 5′ 1stDS₂₂₁-X₂₂₁-X₂₂₂-2ndDS₂₂₁ 3′(1stSS₂₂₁, 2ndSS₂₂₁, X′₂₂₁, X′₂₂₂, DM′ and St_(P221) are the same asdescribed above. 1stDS₂₂₁, 2ndDS₂₂₁ represent the nucleotide sequenceregion which forms complementary double strand with 1stSS₂₂₁, 2ndSS₂₂₁,respectively; X₂₂₁ and X′₂₂₂ represent nucleotide, and X′₂₂₁ and X₂₂₁,and X′₂₂₂ and X₂₂₂ are complementary nucleotides, respectively.).

In the above-described hybrid, DM′-St_(p221) and St_(P221), namely, thenucleotide sequence which is complementary to the deleted nucleotideregion and the probe side stem sequence, are coupled together and formthe stem structure or the loop structure.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₂₂₁-X′₂₂₁-DM′-St_(P221)-X′₂₂₂-2ndSS₂₂₁ 5′Genome: 5′ 1stDS₂₂₁-X₂₂₁-DM-X₂₂₂-2ndDS₂₂₁ 3′(1stSS₂₂₁, 2ndSS₂₂₁, 1stDS₂₂₁, 2ndDS₂₂₁, X′₂₂₁, X′₂₂₂ DM′, and St_(P221)are the same as described above. X₂₂₁ and X₂₂₂ represent nucleotide,respectively; and are complementary to X′₂₂₁ and X′₂₂₂, respectively. DMrepresents the deleted nucleotide region.).

In the above-described hybrid, since the probe side stem sequence formsthe stem structure, the above-described hybrid has the stem structurewhich is designed by the probe.

As mentioned above, in the case where the mutant-type DNA is a DNA whichhas the deleted nucleotide region, and the hybrid which has the stemstructure in the probe side is formed; the hybrid further comprises thehybrid which forms the stem structure or the loop structure in thegenome side. The details will be described in (IV).

(II-2) In the Case where the Hybrid which has the Stem Structure in theGenome Side is Formed

In the case where the mutant-type DNA is the one which has the deletednucleotide region, and at the time when the probe has hybridized, thestem structure is formed in the genome side, and at least the wild-typehybrid forms the stem structure; the probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₂₃₁-2ndSS₂₃₁ 5′(1stSS₂₃₁ represents single-stranded nucleotide sequence which does notcomprise the deleted nucleotide region and which is complementary to the3′-side of the mutant DNA, and 2ndSS₂₃₁ represents single-strandednucleotide sequence which does not comprise the deleted nucleotideregion and which is complementary to the 5′-side of the mutant DNA,respectively.).

The above-described deleted nucleotide region means the nucleotideregion in the wild-type DNA which is defected in the mutant-type DNA.

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₂₃₁-2ndSS₂₃₁ 5′Genome: 5′ 1stDS₂₃₁-2ndDS₂₃₁ 3′(In said hybrid, 1stSS₂₃₁ and 2ndSS₂₃₁ are the same as described above.1stDS₂₃₁, 2ndDS₂₃₁ represent the nucleotide sequence region which formscomplementary double strand with 1stSS₂₃₁, 2ndSS₂₃₁, respectively.) Theabove-described hybrid does not form the stem structure because theprobe and the genome can form the base pairs completely.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₂₃₁-2ndSS₂₃₁ 5′Genome: 5′ 1stDS₂₃₁-DM-2ndDS₂₃₁ 3′(In said hybrid, 1stSS₂₃₁, 2ndSS₂₃₁, 1stDS₂₃₁, and 2ndDS₂₃₁ are the sameas described above. DM represents a sequence which is the deletednucleotide region, and which forms the stem structure.)

In the above-described hybrid, since the deleted nucleotide region formsthe stem structure in the genomic DNA side, said hybrid has the stemstructure.

As mentioned above, in the case where the mutant-type DNA is a DNA whichhas the deleted nucleotide region, and which forms the hybrid having thestem structure in the genome side; the hybrid further includes thehybrid which forms the stem structure or the loop structure in thegenome side. The details will be described in (IV).

(III) In the Case where DNA is a Mutant-Type DNA which has the InsertedNucleotide Region(III-1) in the Case where the Hybrid which has the Stem Structure in theGenome Side is Formed

(a) In the Case where at Least the Mutant-Type Hybrid Forms the StemStructure

In the case where the mutant-type DNA is a DNA which has the insertednucleotide region, and at the time when the probe has hybridized, atleast the mutant-type hybrid which has the stem structure in the genomeside is formed, the probe pertaining to the present invention to be usedhas, for example, the following structure.

3′ 1stSS₃₁₁-X′₃₁₁-X′₃₁₂-2ndSS₃₁₁ 5′(1stSS₃₁₁, 2ndSS₃₁₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₃₁₁ and X′₃₁₂ each represents nucleotide.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₃₁₁-X′₃₁₁-X′₃₁₂-2ndSS₃₁₁ 5′Genome: 5′ 1stDS₃₁₁-X₃₁₁-IM-X₃₁₂-2ndDS₃₁₁ 3′(In the hybrid, 1stSS₃₁₁, 2ndSS₃₁₁, X′₃₁₁ and X′₃₁₂ are the same asdescribed above. 1stDS₃₁₁, 2ndDS₃₁₁ represent the nucleotide sequenceregion which forms complementary double strand with 1stSS₃₁₁ and2ndSS₃₁₁, respectively; X₃₁₁ and X₃₁₂ represent nucleotide,respectively; and X′₃₁₁ and X₃₁₁, and X′₃₁₂ and X₃₁₂ are complementarynucleotides. IM represents a sequence of the inserted nucleotideregion.).

In said hybrid, the sequence of the inserted nucleotide region forms thestem structure.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₃₁₁-X′₃₁₁-X′₃₁₂-2ndSS₃₁₁ 5′Genome: 5′ 1stDS₃₁₁-X₃₁₁-X₃₁₂-2ndDS₃₁₁ 3′(1stSS₃₁₁, 2ndSS₃₁₁, 1stDS₃₁₁, 2ndDS₃₁₁, X′₃₁₁ and X′₃₁₂ are the same asdescribed above. X₃₁₁ and X₃₁₂ represent nucleotide, respectively; andrepresents nucleotide which is complementary to X′₃₁₁ and X′₃₁₂,respectively.) In said hybrid, since the probe and the genome form basepairs completely, the stem structure will not be formed.

(b) In the Case where at Least the Wild-Type Hybrid Forms the StemStructure

In the case where the mutant-type DNA is the one which has the insertednucleotide region, and at the time when the probe has hybridized, thestem structure is formed in the probe side, and at least the wild-typehybrid forms the stem structure (the wild-type hybrid forms a designedstem structure); the probe pertaining to the present invention to beused has, for example, the following structure.

3′ 1stSS₃₂₁-X′₃₂₁-X′₃₂₂-2ndSS₃₂₁ 5′(1stSS₃₂₁, 2ndSS₃₂₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; and X′₃₂₁ and X′₃₂₂ represents nucleotide, respectively.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₃₂₁-X′₃₂₁-X′₃₂₂-2ndSS₃₂₁ 5′Genome: 5′ 1stDS₃₂₁-X₃₂₁-IM-St_(G321)-X₃₂₂-2ndDS₃₂₁ 3′(In said hybrid, 1stSS₃₂₁, 2ndSS₃₂₁, X′₃₂₁, and X′₃₂₂ are the same asdescribed above. 1stDS₃₂₁, 2ndDS₃₂₁ represent the nucleotide sequenceregion which forms complementary double strand with 1stSS₃₂₁ and2ndSS₃₂₁, respectively; X₃₂₁ and X₃₂₂ represent nucleotide, and theseare the nucleotide complementary to X′₃₂₁ and X′₃₂₂, respectively. IMrepresents the sequence of the inserted nucleotide region, and St_(G321)represents the genome side stem sequence.)

In said hybrid, IM-St_(G321) nucleotide chain, namely, the insertednucleotide region and the genome-type hybrid are coupled, and therebythe stem structure or the loop structure is formed.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₃₂₁-X′₃₂₁-X′₃₂₂-2ndSS₃₂₁ 5′Genome: 5′ 1stDS₃₂₁-X₃₂₁-St_(G321)-X₃₂₂-2ndDS₃₂₁ 3′(In said hybrid, 1stSS₃₂₁, 2ndSS₃₂₁, 1stDS₃₂₁, 2ndDS₃₂₁, X′₃₂₁, X′₃₂₂,X₃₂₁, X₃₂₂ and St_(G321) are the same as described above.)

In said hybrid, the genome side stem sequence forms the stem structure.

As mentioned above, in the case where the mutant-type DNA is a DNA whichhas the inserted nucleotide region, and which forms the hybrid havingthe stem structure in the genome side; the hybrid further includes theone which forms the stem structure or the loop structure in the probeside. The details will be described in (IV).

(III-2) In the Case where the Hybrid which has the Stem Structure in theProbe Side is Formed

(a) In the Case where at Least the Wild-Type Hybrid Forms the StemStructure

In the case where the mutant-type DNA is the one which has the insertednucleotide region, and at the time when the probe has hybridized, thestem structure is formed in the probe side, and at least the wild-typehybrid forms the stem structure, the probe pertaining to the presentinvention to be used has, for example, the following structure.

3′ 1stSS₃₂₁-X′₃₂₁-IM′₃₂₁-X′₃₂₂-2ndSS₃₂₁ 5′(1stSS₃₂₁, 2ndSS₃₂₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₃₂₁ and X′₃₂₂ represents arbitrary nucleotide,respectively; IM′₃₂₁ represents the nucleotide sequence which iscomplementary to the inserted nucleotide region, and which forms thestem structure.)

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₃₂₁-X′₃₂₁-IM′₃₂₁-X′₃₂₂-2ndSS₃₂₁ 5′Genome: 5′ 1stDS₃₂₁-X₃₂₁-1M₃₂₁-X₃₂₂-2ndDS₃₂₁ 3′(In the hybrid, 1stSS₃₂₁, 2ndSS₃₂₁, X′₃₂₁, X′₃₂₂, and IM′₃₂₁ are thesame as described above. 1stDS₃₂₁, 2ndDS₃₂₁ represent the nucleotidesequence region which forms complementary double strand with 1stSS₃₂₁and 2ndSS₃₂₁, respectively; X₃₂₁ and X₃₂₂ represent the nucleotides,respectively; and X′₃₂₁ and X₃₂₁, and X′₃₂₂ and X₃₂₂ are complementarynucleotides. IM₃₂₁ represents the sequence of the inserted nucleotideregion.).

In said hybrid, since the probe and the genome form base pairscompletely, the stem structure is not formed.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₃₂₁-X′₃₂₁-IM′₃₂₁-X′₃₂₂-2ndSS₃₂₁ 5′Genome: 5′ 1stDS₃₂₁-X₃₂₁-X₃₂₂-2ndDS₃₂₁ 3′(In the hybrid, 1stSS₃₂₁, 2ndSS₃₂₁, X′₃₂₁, X′₃₂₂, IM′₃₂₁, 1stDS₃₂₁,2ndDS₃₂₁, X₃₂₁, and X₃₂₂ are the same as described above.).

In said hybrid, the nucleotide chain which is complementary to theinserted nucleotide region forms the stem structure.

(b) In the Case where at Least the Mutant-Type Hybrid Forms the StemStructure

In the case where the mutant-type DNA is the one which has the insertednucleotide region, and at the time when the probe has hybridized, thestem structure is formed in the probe side, and at least the mutant-typehybrid forms the stem structure (the mutant-type hybrid forms the stemstructure), the probe pertaining to the present invention to be usedhas, for example, the following structure.

3′ 1stSS₃₃₁-X′₃₃₁-IM′₃₃₁-Stp₃₃₁-X′₃₃₂-2ndSS₃₃₁ 5′(1stSS₃₃₁, 2ndSS₃₃₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; and X′₃₃₁ and X′₃₃₂ represents arbitrary nucleotide,respectively; IM′₃₃₁ represents the nucleotide sequence which iscomplementary to the substituted nucleotide region; and Stp₃₃₁represents the probe side stem sequence.).3′ 1stSS₃₃₁-X′₃₃₁-Stp₃₃₁-IM′₃₃₁-X′₃₃₂-2ndSS₃₃₁ 5′(1stSS₃₃₁, 2ndSS₃₃₁, X′₃₃₁, X′₃₃₂, IM′₃₃₁, and Stp₃₃₁ are the same asdescribed above.).

When the above-described probe is hybridized with the mutant-type DNA,the following mutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₃₃₁-X′₃₃₁-IM′₃₃₁-Stp₃₃₁-X′₃₃₂-2ndSS₃₃₁ 5′Genome: 5′ 1stDS₃₃₁-X₃₃₁-IM′₃₃₁-X₃₃₂-2ndDS₃₃₁ 3′orProbe: 3′ 1stSS₃₃₁-X′₃₃₁-Stp₃₃₁-IM′ 331-X′₃₃₂-2ndSS₃₃₁ 5′Genome: 5′ 1stDS₃₃₁-X₃₃₁-IM₃₃₁-X₃₃₂-2ndDS₃₃₁ 3′(In the hybrid, 1stSS₃₃₁, 2ndSS₃₃₁, X′₃₃₁, X′₃₃₂, and IM′₃₃₁ are thesame as described above. 1stDS₃₃₁ and 2ndDS₃₃₁ represent the nucleotidesequence region which forms complementary double strand with 1stSS₃₃₁and 2ndSS₃₃₁, respectively; X₃₃₁ and X₃₃₂ represent the nucleotides,respectively, and X′₃₃₁ and X₃₃₁, and X′₃₃₂ and X₃₃₂ are complementarynucleotides, respectively. IM₃₃₁ is a sequence of the insertednucleotide region which represents the nucleotide complementary toIM′₃₃₁; and Stp₃₃₁ represents the probe side stem sequence.).

In said hybrid, since the probe side stem sequence forms the stemstructure, said hybrid has the stem structure which is designed in theprobe.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₃₃₁-X′₃₃₁-IM′₃₃₁-Stp₃₃₁-X′₃₃₂-2ndSS₃₃₁ 5′Genome: 5′ 1stDS₃₃₁-X₃₃₁-X₃₃₂-2ndDS₃₃₁ 3′orProbe: 3′ 1stSS₃₃₁-X′₃₃₁-Stp₃₃₁-IM′₃₃₁-X′₃₃₂-2ndSS₃₃₁ 5′Genome: 5′ 1stDS₃₃₁-X₃₃₁-X₃₃₂-2ndDS₃₃₁ 3′(In the hybrid, 1stSS₃₃₁, 2ndSS₃₃₁, X′₃₃₁, X′₃₃₂, IM′₃₃₁, Stp₃₃₁,1stDS₃₃₁, 2ndDS₃₃₁, X₃₃₁ and X₃₃₂ are the same as described above.).

In the above-described mutant-type hybrid, the probe side stem sequenceand the inserted nucleotide region are coupled, and thereby the stemstructure or the loop structure is formed.

As mentioned above, in the case where the mutant-type DNA is a DNA whichhas the inserted nucleotide region, and which forms the hybrid havingthe stem structure in the probe side; the hybrid further includes thehybrid which forms the stem structure or the loop structure in thegenome side. The details will be described in (IV).

(IV) In the Case where the Stem Structure or the Loop Structure isFurther Formed in the Chain Opposite to the Nucleotide Chain which hasthe Stem Structure

In the hybrid pertaining to the present invention described in above (I)to (III), the stem structure or the loop structure, preferably the stemstructure, may be further formed in the chain in the opposite side ofsequence which has the stem structure, namely, it may be formed in theprobe side when the stem structure is present in the genome side, or itmay be formed in the genome side when the stem structure is present inthe probe side, and thereby sharpness (precision) of separation of themutant-type hybrid from the wild-type hybrid can be increased.

In that case, as for the probe, when the stem structure is present inthe genome side, a sequence for forming stem sequence or the loop may befurther inserted in the sequence of the above-described probe, and whenthe stem structure is present in the probe side, to form the stemstructure or the loop structure in the genome, a complementary chaincorresponding to the sequence which forms these structure may be removedfrom the probe.

Specifically, in the case where, for example, the mutant-type DNA is theone which has the inserted nucleotide region, and at the time when theprobe has hybridized, at least the mutant-type hybrid which has the stemstructure in the genome side is formed (corresponding to theabove-described III-1), to form further the stem structure or the loopstructure in the probe side, the probe pertaining to the presentinvention to be used may be, for example, the one which has thefollowing structure.

3′ 1stSS₄₁₁-X′₄₁₁-St_(op)-X′₄₁₂-2ndSS₄₁₁ 5′(1stSS₄₁₁, 2ndSS₄₁₁ represent the single-stranded nucleotide sequencewhich is complementary to the wild-type DNA and the mutant-type DNA,respectively; X′₄₁₁ and X′₄₁₂ represent respective nucleotides; St_(op)represents the probe side stem sequence, or a sequence which forms theloop structure in the probe side.).

When said probe is hybridized with the mutant-type DNA, the followingmutant-type hybrid is formed.

Mutant-Type Hybrid

Probe: 3′ 1stSS₄₁₁-X′₄₁₁-St_(op)-X′₄₁₂-2ndSS₄₁₁ 5′Genome: 5′ 1stDS₄₁₁-X₄₁₁-IM-X₄₁₂-2ndDS₄₁₁ 3′(In the hybrid, 1stSS₄₁₁, 2ndSS₄₁₁, X′₄₁₁, and X′₄₁₂ are the same asdescribed above. 1stDS₃₁₁, 2ndDS₃₁₁ represent the nucleotide sequenceregion which forms complementary double strand with 1stSS₃₁₁ and2ndSS₃₁₁, respectively; X₄₁₁ and X₄₁₂ represent the nucleotides,respectively; and X′₄₁₁ and X₄₁₁, and X′₄₁₂ and X₄₁₂ are complementarynucleotides. IM represents the sequence of the inserted nucleotideregion.).

In said hybrid, on the genome side, the sequence of the insertednucleotide region forms the stem structure, and on the probe side, thesequence which forms the stem structure in the probe side or thesequence which forms the loop structure in the probe side forms the stemstructure or the loop structure.

When the above-described probe is hybridized with the wild-type DNA, thefollowing wild-type hybrid is formed.

Wild-Type Hybrid

Probe: 3′ 1stSS₄₁₁-X′₄₁₁-St_(op)-X′₄₁₂-2ndSS₄₁₁ 5′Genome: 5′ 1stDS₄₁₁-X₄₁₁-X₄₁₂-2ndDS₄₁₁ 3′(1stSS₄₁₁, 2ndSS₄₁₁, 1stDS₄₁₁, 2ndDS₄₁₁, X′₄₁₁, and X′₄₁₂ are the sameas described above.).

In said hybrid, on the probe side, the probe side stem sequence or thesequence which forms the loop structure in the probe side forms the stemstructure or the loop structure.

In addition, as for the case where the stem structure or the loopstructure is intended to be formed in the genome side, for example, inthe case where the mutant-type DNA is the one which has a substitutednucleotide, and at the time when the probe has hybridized, the stemstructure is formed in the probe side (corresponding to theabove-described I-1), and further the stem structure or the loopstructure is intended to be formed in the genome side, the nucleotidechain complementary to the sequence which forms the stem structure orthe loop structure may be removed from the probe side, for example, theprobe in which 4 to 60 mer, preferably 6 to 30 mer, more preferably 6 to20 mer of nucleotide chain coupled to the probe side stem sequence isremoved may be used.

[Method for Detecting Mutant DNA Pertaining to the Present Invention]

The method for detecting mutant DNA of the present invention isperformed by the following steps:

-   (1) A sample containing the single-stranded DNA having substituted    nucleotide, deleted nucleotide region, or inserted nucleotide region    (mutant-type DNA) or/and a wild-type single-stranded DNA    corresponding thereto (wild-type DNA) are contacted with the probe    which is capable of hybridizing with both single-stranded DNA to    form the hybrid with the mutant-type DNA (mutant-type hybrid) or/and    the hybrid with the wild-type DNA (wild-type hybrid);-   (2) Obtained mutant-type hybrid or/and wild-type hybrid are    separated by electrophoresis method; and-   (3) The presence or absence of the mutant-type DNA in the sample is    determined.

As the method in the above-described (1) for forming the mutant-typehybrid or/and the wild-type hybrid by contacting a sample containing themutant-type single-stranded DNA or/and the wild-type DNA with the probe,the probe pertaining to the present invention is added to a solutioncontaining the mutant-type DNA or/and the wild-type DNA so that it makesconcentration in the solution to be 20 nM to 2 μM, preferably 100 nM to500 nM; after that, by carrying out 1 to 4 cycle of reactions, forexample at 90° C. to 100° C. for 2 minutes to 4 minutes (thermaldenaturation), at 30 C to 55° C. for 1 second to 30 seconds (DNAmolecule association reaction), and at 65° C. to 75° C. for 1 minute to4 minutes (template elongation reaction by residual polymerase)(hereinafter, said reaction is sometimes referred to as stem-loop hybridreaction or stem-loop hybrid method), the mutant-type hybrid or/and thewild-type hybrid may be formed. In addition, since the above-describedDNA molecule association reaction in the stem-loop hybrid methodprogresses even under the condition at the time of template elongationreaction by residual polymerase, the stem-loop hybrid method may beperformed by carrying out 1 to 4 cycle of reactions at 90° C. to 100° C.for 2 minutes to 4 minutes (thermal denaturation, DNA moleculeassociation reaction) and at 65° C. to 75° C. for 1 minute to 4 minutes(template elongation reaction by residual polymerase). In addition, inthe stem-loop hybrid reaction, the more the number of cycles, largeramount of the mutant-type hybrid or the wild-type hybrid which is thereaction product can be obtained. However, in four or more cycles, sincethe amount of the product almost remains, usually 2 to 4 cycle ispreferable, and 3 to 4 cycle is more preferable.

More specifically, first, to 20 μL to 40 μL of buffer solution such as,for example, 10 mM to 50 mM Tris buffer solution (pH 8.4 to 9.0) inwhich, for example, 100 ng of mutant-type DNA or/and wild-type DNA isdissolved, the probe pertaining to the present invention is added so asto set the concentration in solution to 20 nM to 2 μM, preferably 100 nMto 500 nM, more preferably 100 nM to 200 nM. Then, for example, bycarrying out 1 to 4 cycle of reactions at 90° C. to 100° C. for 2minutes to 4 minutes (thermal denaturation), at 30° C. to 55° C. for 1second to 30 seconds (DNA molecule association reaction), and at 65° C.to 75° C. for 1 minute to 4 minutes (template elongation reaction byresidual polymerase), the mutant-type hybrid or/and the wild-type hybridare formed.

For the mutant-type single-stranded DNA or the wild-type DNA in theabove-described reaction (1), the one which is made amplified accordingto well-known PCR reaction as mentioned above may be used, and in suchcase, specifically the step of (1) is carried out as follows. That is,for example, 1 pg to 100 pg of DNA to be used as a template is dissolvedin 20 μL to 40 μL of reaction solution; and this solution is furtheradded with, usually 1 pmol to 100 pmol, preferably 1 pmol to 50 pmol ofeach 2 kinds of primers (Forward and Reverse), and usually 4 kinds ofdeoxyribonucleotide triphosphate (dNTPs) so that it provides each 0.01nmol to 50 nmole, preferably 0.1 nmol to 20 nmol; in a buffer solutionsuch as Tris HCl buffer solution containing 1 U to 5 U of thermostableDNA polymerase such as Taq DNA polymerase, KOD DNA polymerase, forexample, a reaction cycle composing, (1) at 93° C. to 98° C. for 10seconds to 10 minutes→(2) 50° C. to 60° C. for 10 seconds to 3minutes→65° C. to 75° C. for 1 minute to 5 minutes is repeated for 30 to40 times; and thus step (1) is performed. After that, to the obtainedreaction solution, 0.1 to 5 times more amount of the probe pertaining tothe present invention than the PCR primer, for example, usually 0.1 pmolto 500 pmol, preferably 0.1 pmol to 50 pmol is added, and the stem-loophybrid reaction is carried out by the same way as described above.

In addition, when mutant-type single-stranded DNA or/and wild-type DNAis amplified by the PCR reaction as described above, the stem-loophybrid reaction can also be performed simultaneously with said PCRreaction. In that case, as for the probe pertaining to the presentinvention, the one which is modified at 3′-terminus and 5′-terminus withphosphate group and the like is utilized, and under the presence of saidprobe, the above-described PCR reaction may be carried out.Specifically, for example, 1 pg to 100 pg of the DNA to be used as atemplate is dissolved in 20 μL to 40 μL of reaction solution; and thissolution is further added with, usually 1 pmol to 100 pmol, preferably 1pmol to 50 pmol of each 2 types of primers (Forward and Reverse), 4kinds of deoxyribonucleotide triphosphate (dNTPs) so that it provideseach usually 0.01 nmol to 50 nmole, preferably 0.1 nmol to 20 nmol, andusually 0.1 nmol to 500 pmol, preferably 0.1 pmol to 50 pmol of theprobe pertaining to the present invention which is modified at3′-terminus and 5′-terminus; and the mixture is reacted in a buffersolution such as 10 to 50m of Tris HCl buffer solution (pH 8.4 to 9.0)containing, for example, 1 U to 5 U of thermostable DNA polymerase, forexample, in a reaction cycle of (1) at 93° C. to 98° C. for 10 secondsto 10 minutes→(2) 50° C. to 60° C. for 10 seconds to 3 minutes→(3) 65°C. to 75° C. for 1 minute to 5 minutes is repeated for 30 to 40 times.

The electrophoresis method in the above-described (2) includes, althoughthere is no limitation specifically so long as it can separate theabove-described mutant-type hybrid from the wild-type hybrid, forexample, electrical separation methods such as electrophoresis methodand dielectrophoresis method comprising isoelectric focusing,SDS-polyacrylamide electrophoresis, agarose gel electrophoresis,acrylamide electrophoresis, capillary electrophoresis, and capillarychip electrophoresis; however, from the points of cooling efficiency,applicable high voltage and separation efficiency, capillaryelectrophoresis, capillary-chip electrophoresis are preferable, andcapillary chip electrophoresis which is suitable for micro-scale sampleanalysis is particularly preferable. It should be noted that, theconditions of these separation methods may be performed according to themethod well-known per se, for example, the capillary-chipelectrophoresis may be performed according to the method described inWO2007/027495, etc.

As for the detection method in the above-described (3), any kind ofdetection method can be used if it is a method well-known per se. Thedetection may be performed by an apparatus such as differentialrefractive index detector, fluorescence detector, UV detector, and amongthem, detection by UV detector, fluorescence detector are preferable,and detection by fluorescence detector is more preferable.

In performing fluorescent detection, various fluorescence detectionmethods may be utilized. Detection may be performed by the method suchas, for example, (I) a method in which after performing the reaction ofthe above-described (1) using a previously fluorescence-labeled probe,fluorescence is detected by carrying out the electrophoresis of (2);(II) a method in which after performing the reaction of (1) using theprobe, electrophoresis is carried out, and after electrophoresis, DNA islabeled by intercalator, etc.

The fluorescence label of the probe employed here includes, for example,cyanine dye. The cyanine dye mentioned here is a compound in which twoheterocyclic rings are coupled through a methine group or a polymethinegroup, and at least one of the heterocyclic rings is nitrogen-containingheterocycle, and the one, in which both of the above-describedheterocyclic rings are nitrogen-containing heterocycle, is preferable.As a substituent group derived from above-described cyanine dye, forexample, a dye derived from Cy-series dye described in U.S. Pat. No.4,981,977, U.S. Pat. No. 5,268,486, U.S. Pat. No. 5,486,616, etc., a dyederived from Dy-series dye described in U.S. Pat. No. 6,083,485, etc., adye derived from HiLyte-series dye described in WO2006/047452, and a dyederived from Alexa-series dye are preferable. In addition, the onederived from commercially available dyes may be utilized, for example,in the case where a dye derived from Cy-series dye is utilized, the onederived from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and so on [all of them aretrade name of Amersham Biosciences]; as for a dye derived from Dy-seriesdye, the one derived from DY-700, DY-701, DY-730, DY-731, DY-732,DY-734, DY-750, DY-751, DY-752, DY-776, DY-780, DY-781, DY-782, and soon; as for a dye derived from HiLyte-series dye, the one derived fromHiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750,and so on (all of them are trade name of AnaSpec Inc.); as for a dyederived from Alexa-series dye, Alexa Fluor Dye 532, Alexa Fluor Dye 546,Alexa Fluor Dye 555, Alexa Fluor Dye 568, Alexa Fluor Dye 594, AlexaFluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye680, Alexa Fluor Dye 700, Alexa Fluor Dye 750, and so on [all of themare trade name of Molecular Probes]; are included as a desirable dye.Among them, a dye derived from Cy-series dye is preferable, among them adye derived from Cy5 is preferable.

In addition, the intercalator employed in the above-described detectionmethod may be the one which emits strong fluorescence by binding withnucleic acid chain, and specifically, acridine dye such as, for example,acridine orange, ethidium compound such as, for example, ethidiumbromide, ethidium homodimer 1 (EthD-1), ethidium homodimer 2 (EthD-2),ethidium bromide monoazide (EMA), and dihydroethidium, iodine compoundsuch as, for example, propidium iodide, hexidium iodide, for example,7-amino actinomycin D (7-AAD), dimeric cyanine series dye such as, forexample, POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3,YOYO-3, TOTO-3 (all of them are trade name of Molecular Probes Inc.),SYTOX series dye such as, for example, SYBR Gold, SYBR Green I and SYBRGreen II, SYTOX Green, SYTOX Blue, and SYTOX Orange (all of them aretrade name of Molecular Probes Inc.), are included. In addition, the onewhich bind to a minor groove in double helix of DNA [for example,4′,6′-diamidine-2-phenylindole, (DAPI: trade name of Molecular ProbesInc.), pentahydrate(bis-benzimide) (Hoechst 33258: trade name ofMolecular Probes Inc.), trihydrochloride (Hoechst 33342: trade name ofMolecular Probes Inc.), bisbezimide dye (Hoechst 34580: trade name ofMolecular Probes Inc.) and so on], the one which bind specifically toadenine-thymine (A-T) sequence [for example, acridine dye such as9-amino-6-chloro-2-methoxyacridine (ACMA),bis-(6-chloro-2-methoxy-9-acridinyl) spermine (acridine homodimer), forexample, hyroxystirbamidine etc.], can also be used in the same way asintercalator.

Specifically, the method for detecting mutant DNA of the presentinvention is carried out, for example, as follows.

That is, in the case where the detection of mutant DNA is carried out,for example, for human genomic DNA derived from cancer patient, thehuman genomic DNA which is extracted and refined by using a commerciallyavailable kit and the like is used as a sample, and the PCR reaction isperformed. The sample for PCR reaction may be prepared, for example, bydissolving each primer for detection of target DNA (Forward and Reverse)usually by 100 to 1000 nM, each 4 kinds of deoxyribonucleotidetriphosphate (dNTPs) usually by 0.1 to 500 nM, and Taq DNA polymerase 1to 5 units in 20 μL of buffer solution such as Tris HCl buffer, and byadding 1 ng to 100 ng of human genomic DNA thereto. As for the PCRreaction, for example, by performing 30 to 40 cycles of a reaction cyclecomposed of (1) 93 to 98° C. for 10 to 30 seconds, (2) 50 to 60° C. for10 to 30 seconds, and (3) 68 to 72° C. for 1 to 3 minutes, the targetsingle-stranded DNA can be amplified. After the PCR reaction, by adding0.1 to 10 times more amount of probe than the primer used in PCR, thehybrid reaction is performed. That is, for example, to the PCR product,the probe pertaining to the present invention is added so as to give afinal concentration of 100 to 500 nM, and the mixture is reacted at 90to 100° C. for 2 to 4 minutes, 30 to 55° C. for 1 to 30 seconds, 65 to75° C. for 1 to 4 minutes, and by repeating the reaction for 1 to 4cycles, the hybrid of DNA of detection target can be obtained. Obtainedsolution is separated by an appropriate separation method, for exampleby capillary chip electrophoresis, and by detecting using a fluorescencedetector etc., the mutant-type DNA can be detected.

Hereinafter, the present invention will be explained in more detail byreferring to Examples, Comparative Examples and so on, however, thepresent invention is not limited thereto in any way.

EXAMPLES Example 1: Detection of Single Nucleotide Substitution MutantDNA of KRAS Gene by the Probe which can Form the Hybrid Having the StemStructure

(1) Preparation of Human Genomic DNA Derived from a Colorectal CancerPatient

The human genomic DNA derived from a colorectal cancer patient wasprepared using QIAGEN QIAamp DNA Mini Kit. A 25 mg of frozen cancertissue was homogenized, then buffer solution attached to the kit wasadded and further proteinase K was added, and lysed completely at 56°C., and after treatment with RNaseA, deproteinization was carried out bybuffer solution attached to the kit, then the centrifuged supernatantwas extracted and refined by a spin column attached to the kit, and 50ng/μL of the human genomic DNA was prepared as a template material.After the obtained purified genomic DNA was amplified by the PCRreaction described in next section (3), the amplified DNA was purifiedby the montage PCR of Millipore Corp. and used as a sample DNA, andusing the primer KRAS-Rv [SEQ ID NO: 15] as a sequence primer, sequencecheck was performed by the same method as described later in SyntheticExample 1 (2), and it was confirmed that single nucleotide substitution(GTT) was present on the codon 12 of KRAS gene.

(2) Preparation of the Probe for Stem-Type Loop-Hybrid Reaction (SLHReaction)

The probe mentioned below which was designed so that the probe bindswith mutant nucleotide and that the loop which is formed at the time ofhybridization will be the stem structure was used as the probe for theSLH reaction (IN-1):

-   -   aaggcctgctgaaaatgactgaatataaacttgtggtagttggagctggtatatatataggcgtaggcaaga        gtgccttgacgatacag [SEQ ID NO: 17]        Oligonucleotide synthesis of the above-described primers and        probe was performed through the use of custom synthesis service        of Sigma-Genosys Inc.

It should be noted that, when said probe is used, it is conceived thatthe stem structure shown in FIG. 16 is formed on the probe.

(3) SLH Reaction of Human Genomic DNA Derived from Colorectal CancerPatient

Using the human genomic DNA derived from colorectal cancer patientprepared in the above-described (1) as a sample, the PCR reaction wascarried out with the use of AccuPrime Taq Polymerase System (a kit forPCR reaction, produced by Invitrogen Corp.). That is, firstly, accordingto the product protocol of attachment to the kit, and using each 2 μL of10 μM primers (KRAS-Fw: aaggcctgctgaaaatgactg [SEQ ID NO: 16] andKRAS-Rv: ggtcctgcaccagtaatatgca [SEQ ID NO: 15]) and 2 μL of PCRreaction buffer of attachment to the kit, 20 μL of reaction liquid forPCR was prepared. Then, 25 ng of the human genomic DNA derived fromcolorectal cancer patient was added and suspended in 20 μL of thereaction liquid for PCR, and used it as a sample for PCR.Oligonucleotide synthesis of the above-described primer and probe wasperformed through the use of custom synthesis service of Sigma-GenosysInc. Using this sample for PCR, and with the use of DNA Thermal Cyclerof MJ Research Inc. (DNA Engine PTC200), 36 cycles of PCR reaction wascarried out under the following reaction condition.

Reaction Condition of the PCR:

Thermals denaturation: 95° C., 15 sec.

Annealing: 55° C., 15 sec.

Polymerization reaction: 68° C., 47 sec.

After termination of the reaction, the SLH probe (ID.=IN-1:aaggcctgctgaaaatgactgaatataaacttgtggtagttggagctggtatatatataggcgtaggcaagagtgccttgacgatacag [SEQ ID NO: 17]) was added to the PCR product so that itgives a final concentration 200 nM, and then with the use of DNA ThermalCycler (DNA Engine PTC200, produced by MJ Research, Inc.), 1 cycle ofreaction was carried out on the following reaction condition.

(SLH-Reaction)

PCR product 4.5 μL SLH probe 0.5 μL of 2 μM stock 105° C.  hot lid 95°C. 2 min. 55° C. 0 to 30 sec. 68° C. 4 min.  4° C. pause

(4) Separation and Detection of the Hybrid Generated by the SLH Reaction(Microchip Electrophoresis)

The reaction product obtained in the above-described (3) was subjectedto the microchip electrophoresis method with the use of Agilent 2100Bioanalyzer systems (Agilent Technologies Inc.). In this electrophoresismethod, Agilent DNA1000 Assay kit (produced by Agilent TechnologiesInc.) which is the dedicated Reagent set was used, and according to theproduct protocol attached to the kit, 1.0 μL of each reaction productswas applied. In addition, for peak analysis after electrophoresis,system attached Agilent 2100 Expert software was utilized, and waveformanalysis and calculation of peak mobility were performed.

The results were shown in FIG. 1.

Comparative Example 1: Detection of Single Nucleotide SubstitutionMutant DNA Of KRAS Gene by the Probe (LH Probe) which can Form theHybrid not Having the Stem Structure

The probe described below which forms the loop structure at the time ofthe hybrid formation was used as the probe for LH reaction (Del-7).

LH probe ID. = Del-7: [SEQ ID NO: 18]aaggcctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagct 

It should be noted that, when said probe is used, it is conceived thatthe loop structure of the structure shown in FIG. 17 is formed on thegenomic DNA.

The human genomic DNA derived from colorectal cancer patient wasmeasured by the same method as Example 1 except for using theabove-described probe. The results were shown in FIG. 2.

As is clear from the results shown in FIG. 1 and FIG. 2, it turned outthat the human genomic DNA derived from colorectal cancer patient whichcould not be separated when the probe which does not form the hybridhaving the stem structure (probe which forms only the loop structure,Del-7 [SEQ ID NO: 18]) was used (FIG. 2), could be separated when theprobe which forms the hybrid having the stem structure (IN-1) [SEQ IDNO: 17] was used (FIG. 1). That is, it turned out that, by giving thestem structure to the hybrid, the capability of discrimination betweenthe mutant-type DNA and the wild-type DNA was markedly improved ascompared with the hybrid having mere loop structure.

Synthetic Example 1: Preparation of Sample Clone Having a Sequence ofMutant-Type DNA and Wild-Type DNA in the KRAS Gene

As for the number of single nucleotide substitution in codon 12 andcodon 13 of the KRAS gene exon 1, there exist 12 kinds of mutations insum total of 3 kinds of mutations in each primary nucleotide andsecondary nucleotide in the codon. And so, by the overlap extension PCRmutagenesis method (BioTechniques, Vol. 48, No. 6, June 2010, pp.463-465) using synthetic oligonucleotides, the wild-type DNA sequencewhich was introduced with these mutant-type DNA was amplified by thePCR, and further, the amplification product was cloned into plasmidusing pGEM-T Vector system (Promega Corp.), and sample clones having asequence of 12 kinds of mutant-type DNA and wild-type DNA were obtained.The details of the procedure of this cloning were presented below.

That is, firstly, according to the protocol attached to QIAGEN QIAamp®DNA Blood Midi Kit, normal human genomic DNA was extracted and refinedas described below from 2 mL of whole blood of normal healthy subject.Namely, to 2 mL of the blood, 200 μL of QIAGEN Protease provided in thekit was added and mixed. Subsequently, after adding 2.4 mL of Buffer ALof kit attachment and mixing completely, the mixture was incubated at70° C. for 10 minutes. Then, after adding 2 mL of ethanol (96 to 100%)to the sample and mixed completely, the obtained solution wastransferred to QIAamp Midi column in the centrifugal tube provided inthe kit. Subsequently, the solution was centrifuged by 1,850×g (3,000rpm) for 3 minutes, the QIAamp Midi column was removed, filtrate wasdiscarded, the removed QIAamp Midi column was returned to the centrifugetube, and then centrifuged by 1,850×g (3,000 rpm) for 3 minutes. Again,the QIAamp Midi column was removed, filtrate was discarded, and theremoved QIAamp Midi column was returned to the 15 ml microcentrifugetube. Subsequently, 2 mL of Buffer AW1 provided in the kit was added tothe QIAamp Midi column, and centrifugal separation was carried out by4,500×g (5,000 rpm) for 1 minute. Further, 2 mL of Buffer AW2 providedin the kit was added to the QIAamp Midi column, and centrifugalseparation was carried out by 4,500×g (5,000 rpm) for 15 minutes. TheQIAamp Midi column was transferred to a new microcentrifuge tubeprovided in the kit, and the tube containing filtrate was removed. Atthe end, at room temperature (15 to 25° C.), 300 μL of Buffer AEprovided in the kit was placed directly on a membrane of QIAamp Midicolumn by pipetting. The lid was closed, and after incubation at roomtemperature for 5 minutes, centrifugal separation was carried out by4,500×g (5,000 rpm) for 2 minutes, and thus, an eluate containing normalhuman genomic DNA was recovered from the whole blood of normal subject.

Subsequently, using the obtained normal human genomic DNA as a template,PCR reactions were carried out using the Platinum Taq polymerase system(Invitrogen Corp., Carlsbad, Calif.). According to the product protocolsupplied with the kit, twelve PCR reactions were prepared by combining 1μL of 10 μM primer 1204 [SEQ ID NO: 1] with 1 μL each of the followingtwelve 10 μM primers: primer 1211, 1212, 1213, 1221, 1222, 1223, 1311,1312, 1313, 1321, 1322, and 1323 (see Table 1 below), with 5 μL of PCRreaction buffer supplied with the kit, 1 μL of 10 mM dNTPs, and 50 ng ofnormal human genomic DNA in 50 μL of the PCR reaction liquid.

TABLE 1 Primer ID Sequence SEQ ID NO: 1211 acttgtggtagttggagctcSEQ ID NO: 2 1212 acttgtggtagttggagctt SEQ ID NO: 3 1213acttgtggtagttggagcta SEQ ID NO: 4 1221 cttgtggtagttggagctgc SEQ ID NO: 51222 cttgtggtagttggagctgt SEQ ID NO: 6 1223 cttgtggtagttggagctgaSEQ ID NO: 7 1311 tgtggtagttggagctggtc SEQ ID NO: 8 1312tgtggtagttggagctggtt SEQ ID NO: 9 1313 tgtggtagttggagctggtaSEQ ID NO: 10 1321 ggagctggtgccgtaggcaag SEQ ID NO: 11 1322gtggtagttggagctggtgt SEQ ID NO: 12 1323 gtggtagttggagctggtgaSEQ ID NO: 13

Using this sample for PCR, the 35 cycles of PCR reaction was carried outfor 12 kinds with the use of DNA Thermal Cycler of MJ Research Inc. (DNAEngine PTC200) on the following reaction condition.

PCR Reaction Condition:

Thermal denaturation: 94° C., 20 sec.

Annealing: 55° C., 20 sec.

Polymerization reaction: 72° C., 20 sec.

After dilution of these 12 kinds of PCR products by 1000 times, toincrease the precision of mutation introducing efficiency, the PCRreaction was further performed using Pwo SuperYield DNA Polymerase(Roche) which is a polymerase with the highest replication accuracy.That is, as for the PCR reaction solution, using 1 μL of each 12 kindsof 10 μM primer pairs for mutation introduction, 5 μL of reaction bufferprovided in the kit, and 1 μL of 10 mM dNTPs, and by adding 0.25 μL of1000 times diluted corresponding mutation introduction PCR productdescribed in the previous section, 50 μL of reaction liquid for PCR wasprepared. Using this sample for PCR, the PCR reaction of 20 cycles wasperformed for the 12 kinds of mutation introduction PCR products on thefollowing reaction condition.

PCR Reaction Condition:

Thermal denaturation: 94° C., 15 sec

Annealing: 55° C., 30 sec.

Polymerization reaction: 68° C., 20 sec.

Thereby, 12 kinds of high-definition mutation introduction PCR productsin which the mutation have been introduced into the codon 12 or thecodon 13 were obtained.Next, in order to amplify DNA which comprises an upstream part of thetarget final PCR product and has an overlapping part with theabove-described mutation introduction PCR product by high-precision PCR,using the normal human genomic DNA as a template, and using PwoSuperYield DNA Polymerase (Roche), and using each 1 μL of 10 μM primer1203 N: gtactggtggagtatttgatagtg [SEQ ID NO: 14] and primer R:ggtcctgcaccagtaatatgca [SEQ ID NO: 15], and using 5 μL of PCR reactionbuffer of attachment to the kit and 1 μL of 10 mM dNTPs; and 25 μL ofnormal human genomic DNA was added and suspended in 50 μL of reactionliquid for PCR and used as a sample for PCR; and the PCR reaction of 30cycles was performed on the following reaction condition.

PCR Reaction Condition:

Thermal denaturation: 94° C., 15 sec.

Annealing: 55° C., 30 sec.

Polymerization reaction: 68° C., 20 sec.

Using 246 bp PCR product for the upstream side (1203N, R) obtained inthis way, and each of 12 kinds of mutation introduction PCR products(162 to 175 bp) obtained in the preceding paragraph, and using PlatinumTaq DNA Polymerase High Fidelity (Invitrogen Corp.), overlap extensionPCR was performed. Reaction solution was made up using 5 μL of attachedPCR reaction buffer, and 1 μL of 10 mM dNTPs, and 2 μL of 50 mM MgSO₄,and each 1 μL of PCR product for upstream side (1203N, R) and mutationintroduction PCR products were added and suspended in 50 μL of reactionliquid for PCR and used as a sample for PCR; and the PCR reaction of 2cycles was performed on the following reaction condition.

PCR Reaction Condition:

Thermal denaturation: 94° C., 20 sec.

Annealing: 50° C., 30 sec.

Polymerization reaction: 68° C., 20 sec.

After termination of this overlap extension PCR reaction, each 1 μL of10 μM primer 1203 N: gtactggtggagtatttgatagtg [SEQ ID NO: 14] and 1204:catgaaaatggtcagagaaacc [SEQ ID NO: 1] was added to the above-describedPCR reaction solution, and the PCR reaction of 25 cycles was performedcontinuously on the following reaction condition.

PCR Reaction Condition:

Thermal denaturation: 94° C., 20 sec.

Annealing: 55° C., 20 sec.

Polymerization reaction: 68° C., 20 sec.

The PCR product corresponding to the wild type was prepared with the useof Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Corp.) byperforming the PCR reaction of 30 cycles on the following reactioncondition using 1 μL of each 10 μM primers 1203N and 1204, 5 μL of kitattached PCR reaction buffer, 1 μL of 10 mM dNTPs, and 2 μL of 50 mMMgSO₄, and as a template, 50 ng of normal human genomic DNA was addedand suspended in 50 μL of reaction liquid for PCR.

PCR Reaction Condition:

Thermal denaturation: 94° C., 20 sec.

Annealing: 55° C., 20 sec.

Polymerization reaction: 68° C., 20 sec.

12 kinds of mutation introduction PCR products of 287 bp and 1 kind ofwild type PCR product which were obtained by a series of these PCRreactions were inserted in plasmid vector pGEM-T easy by TA cloningmethod using pGEM-T Vector System (Promega, Corp.). Ligation reactionwas carried out at room temperature for 1 hour using Rapid LigationBuffer and T4 ligase which were provided in the kit.Then, using E. coli JM109 Competent Cells which was produced by ToyoboCo., Ltd., and according to its product protocol, the transformation ofE. coli JM109 Competent Cells was performed at 42° C. for 45 sec. usingthe above obtained recombinant DNA. After that, the obtainedtransformant was cultured on a plate of LB-agar medium containing 100μg/mL ampicillin, 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG), and40 μg/mL X-Gal at 37° C. for 16 hours. After cultivation, by picking upwhite colonies, the transformant for the respective clones which weretransduced with recombinant DNA inserted with the target DNA fragmentwere obtained. After that, using plasmid extraction kit (QIAprep SpinMiniprep) of Qiagen Corp., extraction and purification process of DNAwas carried out.

Namely, the transformant for each clone which was proliferated overnightin 5 mL of LB liquid medium containing 100 μg/mL ampicillin wascollected by centrifugation; and after bacteriolysis by alkaline method,the lysate was neutralized with acidic potassium acetate solution, andfrom their supernatant solution after centrifugation, the plasmid DNAwas purified with a purification column provided in the kit.

(2) Confirmation of Sequence of 12 Kinds of Mutant DNA and Wild Type DNA

Next, using the sample clones with sequences of 12 kinds of mutant DNAand wild type DNA which were cloned in the above-described (1), sequenceanalysis by the Big Dye Terminator kit (produced by Applied Biosystems,Inc.) was performed in following procedure according to the productprotocol.

Sample DNA (respective clones): 2 μL (100 ng)

T7 promoter primer: 1 μL (5 pmol)

Premix: 8 μL

That is, deionized sterile water was added to the above-describedmixture so that it might make total volume 20 μL, and using DNA thermalcycler (DNA Engine PTC200, produced by MJ Research, Inc.), the sequencereaction of 30 cycle was performed on the following reaction condition.

96° C., 2 min→(96° C., 10 sec→50° C., 5 sec→60° C., min)×25→4° C.

After refining the obtained sequence reaction product with the use ofgel filtration column (produced by Qiagen, Corp.), using a sequencer(BaseStation, produced by MJ Research, Inc.) and according to theprocedure manual, sequence decipherment of all candidate sequences wascompleted. As the result, it was confirmed that sample clones wereproduced possessing the twelve mutant-type DNA sequences and the twowild-type DNA sequences involving codons 12 and 13, shown in Tables 2and 3, respectively.

TABLE 2 Nucleotide substitution, Amino acid Codon 12 mutant-type DNA.substitution Mutant-type 1: KR12_AG AGT, G12S Mutant-type 2: KR12_CGCGT, G12R Mutant-type 3: KR12_TG TGT, G12C Mutant-type 4: KR12_GA GAT,G12D Mutant-type 5: KR12_GC GCT, G12A Mutant-type 6: KR12_GT GTT, G12VWild-type (Wt): Wt GGT

TABLE 3 Nucleotide substitution, Amino acid Codon 13 mutant-type DNAsubstitution Mutant-type 7: KR13_AG AGC, G13S Mutant-type 8: KR13_CGCGC, G13R Mutant-type 9: KR13_TG TGC, G13C Mutant-type 10: KR13_GA GAC,G13D Mutant-type 11: KR13_GC GCC, G13A Mutant-type 12: KR13_GT GTC, G13VWild-type (Wt): Wt GGC

Example 2: Detection of Single Nucleotide Substitution Mutant DNA ofKRAS Gene by Acryl Amide Gel Electrophoresis Method (1) SLH Reaction

Using each sample clone prepared in the above-described SyntheticExample 1 as a sample, the PCR reaction was performed using theAccuPrime Taq DNA Polymerase System (a kit for the PCR reaction,produced by Invitrogen Corp.). That is, firstly, according to theproduct protocol of attachment to the kit, 1.0 μL of each 10 μM primer(KRAS-Fw; aaggcctgctgaaaatgactg [SEQ ID NO: 16] and KRAS-Rv;ggtcctgcaccagtaatatgca [SEQ ID NO: 15]) and the PCR reaction buffers 2.0μL of attachment to a kit were used to prepare 20.0 μL of reactionliquid for PCR. Then each 2 pg of sample clone was added and suspendedin 20 μL of reaction liquid for PCR, and used as a sample for PCR. Inaddition, for the oligo synthesis of the above-described primer andprobe, custom synthesis service of Sigma-Genosys, Inc. was utilized.Using this sample for PCR, the PCR reaction of 30 cycles was performedwith the use of DNA thermal cycler (DNA Engine PTC200, produced by MJResearch Inc.) on the following reaction condition.

Reaction Condition of the PCR:

Thermal denaturation: 95° C., 15 sec.

Annealing: 55° C., 15 sec.

Polymerization reaction: 68° C., 47 sec.

After termination of the reaction, the SLH probe (ID.=IN-1:aaggcctgctgaaaatgactgaatataaacttgtggtagttggagctggtatatatataggcgtaggcaagagtgccttgacgatacag [SEQ ID NO: 17]) was added to the PCR product so that itgives a final concentration 200 nM, and then with the use of DNA ThermalCycler (DNA Engine PTC200, produced by MJ Research Inc.), 1 cycle ofreaction was carried out on the following reaction condition.

(SLH-Reaction)

PCR product 4.5 μL SLH probe 0.5 μL of 2 μM stock 105° C.  hot lid 95°C. 2 min. 55° C. 30 sec. 68° C. 4 min.  4° C. pause

(2) Separation and Detection of the Hybrid Generated by the SLH Reaction(Polyacrylamide Gel Electrophoresis)

Among various SLH reaction products obtained in the above-described (1),the products obtained by using 7 kinds of mutant-type DNA (KR12_CG,KR12_TG, KR12_GA, KR12_GC, KR12_GT, KR13_CG, and KR13_TG) and wild-typeDNA of each 1.5 μL was added with 1.5 μL of gel loading buffer, andelectrophoresis was performed in a non-denaturing 10% polyacrylamidegel. In addition, as a molecular mass marker, 1.5 μL of 100 bp ladderfor size marker (produced by Promega Corp.) was loaded on the same gel,and electrophoresed. The polyacrylamide gel used was a compact gel(Compact gel C10L, produced by ATTO Corp.) of 6 cm×6 cm, andelectrophoresis was carried out by a small electrophoretic equipment(Compact PAGE AE-7300, produced by ATTO Corp.) using Tris-glycine buffersolution (37.5 mM Tris and 288 mM glycine) as a buffer solution forelectrophoresis at room temperature. After the electrophoresis, the gelwas stained 10 minutes with SYBR Green I (TAKARA BIO Inc., F0513), andwashed with water, then using a laser imaging scanner (AmershamBiosciences Corp., STORM 860), detection was performed by excitationwavelength of 450 nm and detection filter 520LP.

In addition, expected stem structure in each hybrid is as follows. InTable 4, a lower case letter represents the nucleotide which is notcomplementary to the genomic DNA, and the underlined part represents thesequence which is assumed to form the stem structure.

TABLE 4 Type Probe SEQ ID of probe Hybrid NO: Wild type ProbeGGTatatatataGGC SEQ ID NO: 31 Genomic DNA CCA         CCG KR12_CG ProbecGTatatatataGGC SEQ ID NO: 32 Genomic DNA CCA         CCG KR12_TG ProbetGTatatatataGGC SEQ ID NO: 33 Genomic DNA CCA         CCG KR12_GA ProbeGaTatatatataGGC SEQ ID NO: 34 Genomic DNA CCA         CCG KR12_GC ProbeGcTatatatataGGC SEQ ID NO: 35 Genomic DNA CCA         CCG KR12_GT ProbeGtTatatatataGGC SEQ ID NO: 36 Genomic DNA CCA         CCG KR13_CG ProbeGGTatatatatacGC SEQ ID NO: 37 Genomic DNA CCA         CCG KR13_TG ProbeGGTatatatatatGC SEQ ID NO: 38 Genomic DNA CCA         CCG

(3) Result

With respect to the sample clones which possess the sequence of 7 kindsof mutant DNA and wild type, the experimental results obtained by theelectrophoresis after the SLH reaction were shown in FIG. 3. Inaddition, lane 1 to 5 in FIG. 3 shows the results of the use of KR12_CG,KR12_TG, KR12_GA, KR12_GC, KR12_GT, respectively, and lane 6 to 7 showsthe results of the use of KR13_CG and KR13_TG, and wt shows the resultsof the use of wild type.

From the result shown in FIG. 3, it turned out that, when separation anddetection of SLH reaction product obtained by the present invention ismeasured with the use of polyacrylamide gels electrophoresis, 5 kinds ofmutant DNA in which the codon 12 are CGT, TGT, GAT, GCT, and GTT,respectively, and 2 kinds of mutant DNA in which the codon13 are CGC andTGC, respectively can be discriminated.

Example 3: Detection of Single Nucleotide Substitution Mutant DNA ofKRAS Gene by Acryl Amide Gel Electrophoresis Method (1) SLH Reaction

The SLH reaction of each sample clone prepared in Synthetic Example 1was carried out by the same method as Example 2 except for using SLHprobe[ID.=IN-4:aaggcctgctgaaaatgactgaatataaacttgtggtagttggagctggttctgcagaaggtgtaggcaagagtgccttgacgatacag (SEQ ID NO: 22)] instead of using SLH probe(ID.=IN-1) in the above-described Example 2 (1).

It should be noted that, when the above-described probe SEQ ID NO: 22 isused, it is conceived that the stem structure shown in FIG. 18 is formedin the probe side.

(2) Separation and Detection of the Hybrid Generated by the SLH Reaction(Polyacrylamide Gel Electrophoresis)

The gel electrophoresis of the SLH reaction product was carried out bythe same method as the above-described Example 2 (2) except for using 12kinds of mutant-type DNA and wild-type DNA as the various kinds of SLHreaction products obtained in the above-described (1).

(3) Result

With respect to the sample clones which possess the sequence of 12 kindsof mutant DNA and wild type, the experimental results obtained by theelectrophoresis after the SLH reaction were shown in FIG. 4. Inaddition, lane 1 to 6 in FIG. 4 shows the results of the use of KR12_AG,KR12_CG, KR12_TG, KR12_GA, KR12_GC, KR12_GT, respectively, and lane 6 to12 shows the results of the use of KR13_AG, KR13_CG, KR13_TG, KR13_GA,KR13_GC, KR13_GT, and wt shows the results of the use of the wild type.

From the result shown in FIG. 4, it turned out that, when separation anddetection of SLH reaction product obtained by the present inventionusing the probe of the above-described IN-4 is performed bypolyacrylamide gel electrophoresis, 6 kinds of mutant DNA in which thecodon 12 are AGT, CGT, TGT, GAT, GCT, and GTT, respectively, and 6 kindsof mutant DNA in which the codon 13 are AGC, CGC, TGC, GAC, GCC, andGTC, respectively can also be discriminated.

Example 4: Detection of Various Kinds of Single Nucleotide SubstitutionMutant DNA in the KRAS Gene (Microchip Electrophoresis Method)

The SLH reaction product obtained in the above-described Example 2 (1)was subjected to microchip electrophoresis method using Agilent2100Bioanalyzer System (Agilent Technologies Inc.). In the presentelectrophoresis method, Agilent DNA1000 Assay kit (produced by AgilentTechnologies Inc.) which is a reagent kit for exclusive use was used,and according to the product protocol of attachment to the kit, 1.0 μLof each reaction product was applied. In addition, for the peak analysisafter electrophoresis, Agilent2100 expert software attached to thesystem was used, and waveform analysis and calculation of peak mobilitywere performed.

The results are shown in FIG. 5. In addition, the peak number in FIG. 5expresses the product by the following probe: 1 expresses KR12_AG, 2expresses KR12_CG, 3 expresses KR12_TG, 4 expresses KR12_GA, 5 expressesKR12_GC, 6 expresses KR12_GT, 7 expresses KR13_AG, 8 expresses KR13_CG,9 expresses KR13_TG, 10 expresses KR13_GA, 11 expresses KR13_GC, and 12expresses KR13_GT, respectively.

As is clear from the result shown in FIG. 5, when the detection wasperformed by microchip electrophoresis using the SLH reaction of thepresent invention, each peak of the mutant DNA which has mutation on acodon 12 and a codon 13 had separated from the peak of the wild-typeDNA, and it turned out that the mutation was completely discriminable asa mutant-type DNA. That is, this result showed that mutant-type DNAobtained by the SLH reaction was easily detectable with microchipelectrophoresis. The stem structure might be changed by generation ofheat, however, it was considered that, because the electrophoresis wasperformed by microchip electrophoresis method with little generation ofheat, stable structure could be maintained, and this made separationeasy. Furthermore, it was shown that the microchip electrophoresis wassuitable for detection of mutant-type DNA employing such SLH reactionsince it is easy for peak analysis as compared with the gelselectrophoresis.

In addition, like the KRAS gene testing, when mutation occurred in theadjacent codon (for example, codons 12 and 13), the mutations in bothadjacent codons were needed to be detected. However, by the conventionalloop hybrid method using the probe which forms only the loop structure(Del-7, etc.), all of the mutations in the adjacent codons could not bedetected.

As is clear from the results shown in FIG. 5, by the method devised bythe present inventors which employs the hybrid having the stemstructure, it becomes possible to detect the mutations which may begenerated in the adjacent codons 12 and 13 by the same measurementsystem with high precision.

Example 5: Detection of Mutant-Type DNA Having the Insertion NucleotideRegion of UGT1A1

The UGT1A1 gene has a promoter that contains a dinucleotide repeat:(TA)₆. (Referred to as (TA6)). A mutated form of the promoter containsan extra “TA” in the dinucleotide repeat sequence. Thus, the mutationvariant has an expanded dinucleotide repeat: (TA)₇. (Referred to as(TA7)). In this example, a probe is designed that will bind to thewild-type or the mutation variant form of the UGT1A1 gene and induce theformation of a stem structure in the genomic DNA strand.

(1) Human Blood-Derived Genomic DNA and its Preparation

Multiple samples derived from human blood were prepared, and accordingto QIAGEN QIAamp DNA Blood Midi Kit, after treating 2 mL of each wholeblood with proteinase K at 70° C. for 10 minutes, ethanol was added, andthe supernatant solution after centrifugation was extracted and purifiedby QIAamp Midi column, and human genomic DNA was prepared. Using 50 ngof the genomic DNA as a template material, the sequence was determinedby the same method as performed in Synthetic Example 1 (2). Three kindsof sample genotypes were identified: homozygous wild-type DNA (TA6), theheterozygous form with both the wild-type DNA (TA6) and mutantvariant-type DNA (TA7), and the homozygous variant-type DNA (TA7), andused as samples.

(2) Preparation of the Probe for SLH Reaction

The following probe designed so that the stem structure might be formedon the genome sequence at the time of the hybrid formation was employedas the probe for SLH reaction (UGT1Adel-12F:

ctttgtggactgacagctttttatagtcacgtgacacagtcaaacattaacttggtgtatcgattggtttttgatataagtaggagagggcgaac [SEQ ID NO: 21]).

It should be noted that, when said probe [SEQ ID NO: 21] was used, itwas conceived that the following stem structures formed in the genomicDNA strand amplified from the human blood sample that hybridized withprobe [SEQ ID NO: 21], when subjected to the SLH reaction.

When the wild-type DNA (TA6) allele was present, the (TA6) portion ofthe gene formed the stem structure shown in FIG. 19.When mutant-type DNA allele (TA7) was present, the (TA7) portion of thegene formed the stem structure shown in FIG. 20.

(3) SLH Reaction

Using three kinds of samples prepared in the above-described (1), thePCR reaction was performed using the AccuPrime Taq DNA Polymerase System(kit for the PCR reaction, produced by Invitrogen Corp.). That is,firstly, according to the product protocol of attachment to the kit, 1.0μL of each 10 μM primer (ctttgtggactgacagctttttatag [SEQ ID NO: 19] andgctgccatccactgggatc [SEQ ID NO: 20]) and 2.0 μL of the PCR reactionbuffer of attachment to the kit were used to prepare 20.0 μL of reactionliquid for PCR. Then, each 1 ng of human genomic DNA derived from normalhuman blood was added and suspended in 20 μL of reaction liquid for PCR,and used as a sample for PCR. In addition, for the oligo synthesis ofthe above-described primer and probe, custom synthesis service ofSigma-Genosys, Inc. was utilized. Using this sample for PCR, the PCRreaction of 36 cycles was performed with the use of DNA thermal cycler(DNA Engine PTC200, produced by MJ Research Inc.) on the followingreaction condition.

Reaction Condition of the PCR:

Thermal denaturation: 95° C., 15 sec.

Annealing: 55° C., 15 sec.

Polymerization reaction: 68° C., 47 sec.

After termination of the reaction, the SLH probe ID.=UGT1Adel-12F wasadded so that it gives a final concentration 200 nM, and then with theuse of DNA Thermal Cycler of MJ Research Inc. (DNA Engine PTC200), 1cycle of reaction was carried out on the following reaction condition.

(SLH-Reaction)

PCR product 4.5 μL SLH probe 0.5 μL of 2 μM stock 105° C.  hot lid 95°C. 2 min. 55° C. 30 sec. 68° C. 4 min.  4° C. pause

(4) Separation and Detection of the Hybrid Generated by the SLH Reaction(Microchip Electrophoresis)

The SLH reaction product (hybrid) obtained in the above-described (3)was subjected to the microchip electrophoresis method with the use ofAgilent 2100 Bioanalyzer systems (Agilent Technologies Inc.). In thiselectrophoresis method, Agilent DNA1000 Assay kit (produced by AgilentTechnologies Inc.) which is the dedicated reagents was used, andaccording to the attached product protocol, 1.0 μL of each reactionproducts was applied. In addition, for peak analysis afterelectrophoresis, system attached Agilent 2100 Expert software wasutilized, and waveform analysis and calculation of peak mobility wereperformed.

The obtained results were shown in FIG. 6. In addition, in the figure,left panel shows the measurement result of the hetero of the wild-typeDNA (TA6) and mutant-type DNA (TA7), middle panel shows the result ofthe homo of mutant-type DNA (TA7), and the right panel shows the resultof the homo of the wild-type DNA (TA6), respectively.

As is clear from the result shown in FIG. 6, it turned out thataccording to the method of the present invention, discrimination anddetermination of genotype of gene polymorphism *28 of UGT1A1 genepromoter is enabled. That is, it turned out that even if it was amutant-type DNA having an inserted nucleotide, the separation anddetection of the wild-type DNA and the mutant-type DNA could be achievedby the method of the present invention. In addition, when the detectionis a mutant gene which may form such hetero- and homozygosity, it wascommonly difficult to detect the presence or absence of mutation andalso to detect these genotypes with distinction, but it was confirmedthat by employing the method of the present invention, the genotypeswhether it is hetero- or homozygous could be discriminated clearly.

It should be noted that, with respect to UGT1A1 gene, in addition to theabove-mentioned polymorphism *28, detection of polymorphism *6 of exon 1will also be important. By subjecting the SLH reaction solutions of *6and *28 to the same electrophoresis through the use of the method of thepresent invention, type determination of these polymorphism can beperformed at the same time. Namely, fluorescent SLH probe for eachpolymorphism *6 and *28 were prepared, then hybridized with each *6 and*28, and the hybridized materials were mixed. By using this mixture as asample for electrophoresis, only the interactant with SLH probe wasdetected specifically by the fluorescence of the probe and by setting acondition where these hybrids may separate by different mobility,discrimination of each genotype of respective polymorphism can beachieved by single trial.

Example 6: Specific Detection of LH Band for a Variant in UGT1A1Polymorphism *28

The UGT1A1 gene may also be present in additional mutation variantforms. In one mutation variant form, a TA dinucleotide is deleted. Thepromoter contains one fewer dinucleotide repeat than the normal form:(TA)₅. (Referred to as (TA5)). Another mutated form of the promotercontains two extra “TA” dinucleotide sequences. Thus, the mutationvariant has an expanded dinucleotide repeat: (TA)₈. (Referred to as(TA8)). In this example, a probe is designed that will bind to thewild-type or the mutation variant forms of the UGT1A1 gene and inducethe formation of a stem structure in the genomic DNA strand.

(1) Preparation of Sample DNA which has a Sequence of Mutant-Type DNAand Wild-Type DNA in UGT1A1 Gene

Using samples from 200 persons, screening was performed, and thewild-type DNA (TA6), as well as the mutant-type DNA in which withrespect to wild type (TA6), locus *28 has variant alleles such as (TA5)(an allele in which one TA repeat is deleted from the wild-type allele),(TA7) (an allele in which one TA repeat was inserted in the wild-typeallele), and (TA8) (an allele in which two TA repeats were inserted inthe wild-type allele) were obtained and used as sample DNA.

Specifically, screening was carried out as follows. That is, the PCRreaction was carried out for each genomic DNA sample (5 ng) obtainedfrom the samples derived from 200 persons in which locus *28 isheterozygous with one of those variant alleles using AccuprimeTaq(reaction condition: 95° C., 15 sec.; 55° C., 15 sec.; and 68° C., 47sec., for 36 cycles). The obtained PCR amplification product wasincorporated into TA cloning vector pCR2.1TOPO using TOPO® TA CloningSystem (Invitrogen Corp.), and introduced into E. coli One Shot® TOP10(Invitrogen Corp.) by electroporation. Then, the transfected E. coli wascultured in LB agar medium containing kanamycin (25 ng/mL) at 37° C. for18 hours, and the colonies which acquired antibiotics resistance wereselected as an indicator, and the obtained colony was cloned at random.

One loopful E. coli of each colony was cultured in 50 μL of liquid LBculture-medium containing 25 ng/mL ampicillin at 37° C. for 2 to 4hours, and centrifuged. The bacterial precipitate after centrifugationwas suspended by adding 8 μL of Green solution of Clone Checker(Invitrogen Corp.), and then DNA was released from fungal body by theheat shock at 98° C. for 30 seconds. This DNA solution was dispersed in100 μL of distilled water, and it was amplified by TempliPhi (GEHealthcare), and then, sequence of the plasmid DNA was determined bySanger method. By this screening, wild-type DNA (TA6), mutant-type DNA(TA5), mutant-type DNA (TA7), and mutant-type DNA (TA8) were obtained,and used as sample DNA.

(2) Preparation of the Probe for SLH Reaction

The following probe (UGT1Adel-8F), which was designed so that the stemstructure could be formed on a genome sequence at the time of the hybridformation, was synthesized.

ctttgtggactgacagctttttatagtcacgtgacacagtcaaacattaacttggtgtatcgattggtttttgatatatataagtaggagagggcgaac [SEQ ID NO: 23]When electrophoretic mobility of the hybrid was determined by detectingfluorescence of SYBRGreen I (SYBRGreen I), said probe (hereinafter,abbreviated as the probe 1) was used as the probe for SLH reaction. Inaddition, when electrophoretic mobility of the hybrid was determined bydetecting fluorescence of Cy5, the one in which 5′-terminal of thesequence of aforementioned probe was modified with Cy5 (hereinafter,abbreviated as the probe 2) was used as the probe for SLH reaction. Itshould be noted that, as for fluorescent modification, custom synthesisservice of Sigma-Genosys, Inc. was utilized.

When the above-described probes 1 and 2 were used, it was conceived thatthe following stem structures formed in the genome DNA strand amplifiedfrom the human blood sample that hybridized with probe [SEQ ID NO: 23],when subjected to the SLH reaction.

When mutant-type DNA allele (TA5) was present, the (TA5) portion of thegene formed the stem structure shown in FIG. 21.When the wild-type DNA (TA6) allele was present, the (TA6) portion ofthe gene formed the stem structure shown in FIG. 22.When mutant-type DNA allele (TA7) was present, the (TA7) portion of thegene formed the stem structure shown in FIG. 23.When mutant-type DNA allele (TA8) was present, the (TA8) portion of thegene formed the stem structure shown in FIG. 24.

(3) SLH Reaction

Using the four kinds of sample DNA prepared in the above-described (1),the PCR reaction for each sample was performed using the AccuPrime TaqDNA Polymerase System (a kit for the PCR reaction, produced byInvitrogen Corp.).

That is, first, according to the product protocol of attachment to thekit, 1.0 μL of each 10 μM primer (ctttgtggactgacagctttttatag [SEQ ID NO:19] and gctgccatccactgggatc [SEQ ID NO: 20]) and 2.0 μL of the PCRreaction buffer of attachment to the kit were used to prepare 20.0 μL ofreaction liquid for PCR. Then, 1 ng of DNA sample was added andsuspended in 20 μL of reaction solution for PCR, and used as a samplefor PCR. It should be noted that, as for the oligo synthesis of theabove-described primer and probe, custom synthesis service ofSigma-Genosys, Inc. was utilized. Using this sample for PCR, the PCRreaction of 36 cycles was performed with the use of DNA thermal cyclerof MJ Research Inc. (DNA Engine PTC200) on the following reactioncondition.

Reaction Condition of the PCR:

Thermal denaturation: 95° C., 15 sec.

Annealing: 55° C., 15 sec.

Polymerization reaction: 68° C., 47 sec.

After termination of the reaction, the SLH probe 1 was added to each 4kinds of obtained PCR products so that it gives a final concentration200 nM, and then with the use of DNA Thermal Cycler of MJ Research Inc.(DNA Engine PTC200), 1 cycle of SLH reaction was carried out on thefollowing reaction condition.

(SLH-Reaction:)

PCR product 4.5 μL SLH probe 0.5 μL of 2 μM stock 105° C.  hot lid 95°C. 2 min. 55° C. 30 sec. 68° C. 4 min.  4° C. pause

In addition, in the same manner as above, the SLH probe 2 was added toeach 4 kinds of obtained PCR products so that it gives a finalconcentration 200 nM, and 1 cycle of SLH reaction was carried out on theabove-described reaction condition.

(4) Separation and Detection of the Hybrid Generated by the SLH Reaction(Polyacrylamide Gel Electrophoresis)

To 1.5 μL of each 4 samples of the SLH reaction products (hybrid)obtained in the above-described (3) by using SLH probe 1, 1.5 μL of gelloading buffer was added respectively, and electrophoresis was performedin non-denaturing 10% polyacrylamide gel. In addition, as a molecularmass marker, 1.5 μL of 100 bp ladder for size marker (produced byPromega Corp.) was used and loaded on the same gel, and electrophoresed.The polyacrylamide gel used was a compact gel (Compact gel C10L,produced by ATTO Corp.) of 6 cm×6 cm, and electrophoresis was carriedout by a small electrophoretic equipment (ATTO, AE-7300 Compact PAGE)using Tris-glycine buffer solution (37.5 mM Tris, 288 mM glycine) as abuffer solution for electrophoresis at room temperature. After theelectrophoresis, gel was stained with SYBR Green I (TAKARA BIO Inc.,F0513) for 10 minutes and washed with water, and then detection(excitation wavelength 635 nm, detection filter 650LP) was performedusing a laser imaging scanner (Amersham Biosciences Corp., STORM 860).In this occasion, for the fluorescence detection of SYBR Green I,excitation wavelength of 450 nm, detection filter 520LP was utilized.The results obtained were shown in the left panel of FIG. 7. Inaddition, lane 1 shows the result obtained by use of mutant-type DNA(TA5); lane 2 shows the result obtained by the use of mutant-type DNA(TA6); lane 3 shows the result obtained by the use of mutant-type DNA(TA7); lane 4 shows the result obtained by the use of mutant-type DNA(TA8), respectively.

In addition, in the similar manner as above, using 1.5 μL of each 4samples of the SLH reaction products (hybrid) obtained in theabove-described (3) by using SLH probe 2, the electrophoresis wasperformed by the same way as described above, after that, fluorescencedetection (excitation wavelength 635 nm, detection filter 650LP) wasperformed directly as it was. The results obtained were shown in theright panel of FIG. 7. It should be noted that, lane 1 expresses theresult obtained by use of mutant-type DNA (TA5); lane 2 expresses theresult obtained by use of wild-type DNA (TA6); lane 3 expresses theresult obtained by use of mutant-type DNA (TA7); lane 4 expresses theresult obtained by use of mutant-type DNA (TA8), respectively.

When the detection was performed by SYBR Green I (FIG. 7, left panel),the PCR band and LH band could also be confirmed, but other than thesebands, other bands by the nonspecific amplification products which wereassumed to emerge due to TA reiterated sequence were also seen. On theother hand, when detection was performed by Cy5 (FIG. 7, right panel),only LH probe and loop hybrid were seen predominantly. This was assumedthat since the LH probe modified with Cy5 was employed, theamplification products which were not involve in the LH reaction wereundetectable and only the one which hybridized with the LH probe weredetected specifically. Thus, it turned out that by using the probemodified directly by coupling with a fluorescent group such as Cy5, morespecific detection could be performed.

In addition, as is clear from the results shown in FIG. 7, it alsoturned out that, according to the method of the present invention, theLH band for each wild-type DNA (TA6), mutant-type DNA (TA5), (TA7), and(TA8) were detected on the different position, therefore, these allelescould be differentiated in a single electrophoretic assay.

Example 7: Detection of Mutant-Type DNA Having the Insertion NucleotideRegion of UGT1A1 (Effect of Increased Times of LH Cycles on Amount ofLoop Hybrid) (1) Human Blood-Derived Human Genomic DNA and itsPreparation

A 2 ml of human whole blood was processed by proteinase K at 70° C. for10 minutes using QIAamp DNA Blood Midi Kit (QIAGEN Inc.). After that,ethanol was added thereto, and the supernatant solution aftercentrifugation was extracted and purified by QIAamp Midi column, andused it as a sample DNA.

A 50 ng sample of DNA was used as a template material, and the sequencedetermination was performed by the same method as performed in Syntheticexample 1. As a result, it was confirmed that the sample washeterozygous for the UGT1A1 gene, having a wild-type DNA allele (TA6)and mutant-type DNA allele (TA7).

(2) Preparation of the Probe for SLH Reaction

The same probe as described Example 6 (2) was used. That is, the probe 1was used for detecting fluorescence of SYBRGreen I (SYBRGreen I), andthe probe 2 was used for detecting fluorescence of Cy5.

In addition, when said probes were used, it was conceived that thefollowing stem structures formed in the DNA amplified from the humanblood sample that hybridized with probe 1 or probe 2.

When the wild-type DNA allele was present, the (TA6) portion of the geneformed the stem structure shown in FIG. 25.When mutant-type DNA allele (TA7) was present, the (TA7) portion of thegene formed the stem structure shown in FIG. 26.

(3) SLH Reaction

Using the sample DNA prepared in the above-described (1), the PCRreaction was performed in the same way as described in Example 6 (3).

After termination of the reaction, the SLH probe 1 was added to theobtained PCR product so that it gives a final concentration 200 nM, andthen with the use of DNA Thermal Cycler of MJ Research Inc. (DNA EnginePTC200), 1 to 4 cycles of SLH reaction was carried out on the followingreaction condition.

(SLH-Reaction:)

PCR product 4.5 μL SLH probe 0.5 μL of 2 μM stock 105° C.  hot lid 95°C. 2 min. 55° C. 30 sec. 68° C. 4 min.

In addition, in the same manner as above, the SLH probe 2 was added tothe obtained PCR product so that it gives a final concentration 200 nM,and 1 to 4 cycles of SLH reaction was carried out on the above-describedreaction condition.

(4) Separation and Detection of the Hybrid Generated by the SLH Reaction(Polyacrylamide Gel Electrophoresis)

The SLH reaction products (hybrid) which were obtained in theabove-described (3) by 1 to 4 cycles of the SLH reaction using the SLHprobe 1 and PCR reaction product (0 cycle of SLH reaction) wereprepared; and to 1.5 μL of each of them, 1.5 μL of gel loading bufferwas added respectively, and electrophoresis was performed in anon-denaturing 10% polyacrylamide gel. As a molecular mass marker, 1.5μL of 100 bp ladder for size marker (produced by Promega Corp.) wasused, and loaded on the same gel and electrophoresed. The polyacrylamidegel used was a compact gel (Compact gel C10L, produced by ATTO Corp.) of6 cm×6 cm, and electrophoresis was carried out by a smallelectrophoretic equipment (ATTO, AE-7300 Compact PAGE.) usingTris-glycine buffer solution (37.5 mM Tris, 288 mM glycine) as a buffersolution for electrophoresis at room temperature. After theelectrophoresis, the gel was stained for 10 minutes with SYBR Green I(TAKARA BIO Inc., F0513), and washed with water, then detection wasperformed using a laser imaging scanner (Amersham Biosciences Corp.,STORM 860). In this occasion, for the fluorescence detection of SYBRGreen I, excitation wavelength of 450 nm, detection filter 520LP wasutilized. The result obtained was shown in the right panel of FIG. 8. Itshould be noted that, lane 0 expresses the result obtained by the use ofPCR reaction product, lane 1 to 4 expresses each result obtained by 1 to4 cycles of SLH reaction.

In addition, in the similar manner as above, the SLH reaction products(hybrid) which were obtained in the above-described (3) by 1 to 4 cyclesof the SLH reaction using the SLH probe 2 and PCR reaction product (0cycle of SLH reaction) were prepared; and the electrophoresis wasperformed by the same way as described above, after that, fluorescencedetection (excitation wavelength 635 nm, detection filter 650LP) wasperformed directly as it was. The result obtained was shown in the leftpanel of FIG. 8. In addition, lane 0 expresses the result obtained bythe use of PCR reaction product, lane 1 to 4 expresses each resultobtained by 1 to 4 cycles of SLH reaction.

In the left figure, the bands of SLH probe (ss LH-probe in the figure)and the loop hybrid (LH-bands in the figure) were seen remarkably, andit turned out that the amount of monomeric SLH probe was decreased whilethe amount of loop hybrids was increased with the increased times of SLHreaction cycles. Moreover, it was also confirmed that (TA6) and (TA7)appeared as separate bands (in LH-bands in the figure, the upper bandwas (TA6) and the lower band was (TA7)). In addition, in the results ofelectrophoresis of the hybrid obtained by 2 to 4 times of LH cycles, anew band was seen in slightly upward of the band of unbound LH probe,this was supposed to be a homoduplex which was created in the repeatedSLH reaction using the primer region attached to the LH probe byelongation reaction with Taq polymerase along the genomic DNA template.Also, in the detection by SYBR Green I, it turned out that the amount ofloop hybrid (LH-bands in the figure) was increased by increasing numberof cycle of LH reaction. it turned out that in response to increase innumber of times of LH cycle, the amount of loop hybrid (LH-bands in thefigure) increased; and it was also confirmed that (TA6) and (TA7)appeared as separate bands.

Thus, as is clear from the above-described result, it turned out thatthe amount of loop hybrids was increased by increasing times of the LHreaction. It turned out that this was effective in a system with lowloop hybrid formation rate, and especially, by carrying out 3 to 4times, the hybrid can be obtained effectively. It should be noted that,although not shown in the figure, the result of 5 times of LH cycle wasthe almost same result as that of 4 times.

Example 8: Detection of Poly-T Chain Length Polymorphism in NucleotideSequence of TOMM40 Gene (Separation and Detection by the Hybrid Havingthe Stem Structure in the Both Side)

(1) Synthesis of Sample DNA Having a Fixed Poly-T Chain LengthCorresponding to Various Poly-T Chain Length Polymorphisms in rs10524523Marker Sequence of TOMM40 Gene

As a model nucleotide sequence of poly-T chain length polymorphism(rs10524523) in TOMM40 gene, six kinds of sample DNA in the followingTable 5 were synthesized. In addition, Poly-T in the table correspondsto the chain length of poly-A.

TABLE 5 Sample DNA Sequence SEQ ID NO: Q10Ataggcattcgaagccagcccgggcaacatggtgagaccc [SEQ ID NO: (poly-T = 10)catctcAAAAAAAAAAgccagatgcaatggctcat 24] g Q15Ataggcattcgaagccagcccgggcaacatggtgagaccc [SEQ ID NO: (poly-T = 15)catctcAAAAAAAAAAAAAAAgccagatgca 25] atggctcatg Q20Ataggcattcgaagccagcccgggcaacatggtgagaccc [SEQ ID NO: (poly-T = 20)catctcAAAAAAAAAAAAAAAAAAAAgc 26] cagatgcaatggctcatg Q25Ataggcattcgaagccagcccgggcaacatggtgagaccc [SEQ ID NO: (poly-T = 25)catctcAAAAAAAAAAAAAAAAAAAAA 27] AAAAgccagatgcaatggctcatg Q30Ataggcattcgaagccagcccgggcaacatggtgagaccc [SEQ ID NO: (poly-T = 30)catctcAAAAAAAAAAAAAAAAAAAAA 28] AAAAAAAAAgccagatgcaatggctcatg Q35Ataggcattcgaagccagcccgggcaacatggtgagaccc [SEQ ID NO: (poly-T = 35)catctcAAAAAAAAAAAAAAAAAAAAA 29] AAAAAAAAAAAAAAgccagatgcaatggctc atg

(2) Preparation of the Probe for SLH Reaction

The following probe (cy5delTINGC) which was designed to take the loopstructure reflecting the number of nucleotide of the poly-T chain lengthon a genome sequence, and yet to take the stem structure on the probe inthe hybrid formed by SLH reaction with target sequence was synthesized.

gacctcaagctgtcctcttgccccagccctccaaagcattgggattactggcatgagccattgcatctggacgcgcgtagatggggtctcaccatg [SEQ ID NO: 30]

In addition, in order to determine the hybrid by detecting fluorescenceof Cy5, 5′-terminal of said probe sequence was fluorescence modifiedwith Cy5 and employed as the probe for a SLH reaction. It should benoted that, as for fluorescent modification, custom synthesis service ofSigma-Genosys, Inc. was utilized.

When aforementioned probe [SEQ ID NO: 30] was used, it was conceivedthat, by forming the hybrid with target genome sequence, such as any of[SEQ ID NO: 24-29], the stem-loop structure of the following structureshould be formed (see scheme in FIG. 27). Thus, (N) in the scheme ofFIG. 27 may represent different numbers of A nucleotides depending onthe chain length of poly-T polymorphism. For example, if the genomicstrand in the hybrid shown in the scheme of FIG. 27 has the sequence of[SEQ ID NO: 24], (N) represents two A nucleotides between the eight Anucleotides shown, and if the genomic strand's sequence was [SEQ ID NO:29], then (N) represents twenty-seven A nucleotides.

(3) SLH Reaction

Using six kinds of sample DNA prepared in the above-described (1) asrespective template DNA, and using the above-described cy5delTINGC asthe probe for SLH, 4 cycles of the SLH reaction was performed.

Namely, to each 1.0 μL of 5 μM sample DNA (500 nM as concentration inreaction solution), respective 1.0 μL of 2 μM SLH probe (200 nM asconcentration in reaction solution) was added, and after that, 0.04 μLof AccuPrime Taq DNA Polymerase High Fidelity (produced by InvitrogenCorp.), 1.0 μL of product attached 10× buffer, and 2.96 μL of dd H₂Owere added respectively, and thus six kinds of respective 10 μL ofreaction solutions were prepared. Next, using six kinds of reactionsolution, the SLH reaction of 4 cycles was performed with the thermalcycler of MJ Research Inc. (DNA Engine PTC200) on the following reactioncondition.

(SLH Reactions Cycling Conditions)

105° C.  hot lid 95° C.  2 min. 55° C. 15 sec. 68° C.  4 min.

(4) Separation and Detection of the Hybrid Generated by the SLH Reaction(Polyacrylamide Gel Electrophoresis)

To 1.5 μL of each 5 samples of the SLH reaction products (hybridproduct) obtained in the above-described (3) by using SLH probe, 1.5 μLof gel loading buffer was added respectively, and electrophoresis wasperformed in non-denaturing 10% polyacrylamide gel. In addition, as amolecular mass marker, 1.5 μL of 100 bp ladder for size marker (producedby Promega Corp.) was used and loaded on the same gel, andelectrophoresed. The polyacrylamide gel used was C-PAGEL (C10L, producedby ATTO Corp.) of 6 cm×6 cm, and electrophoresis was carried out by asmall electrophoretic equipment (ATTO, AE-7300 Compact PAGE.) usingTris-glycine buffer solution (37.5 mM Tris, 288 mM glycine) as a buffersolution for electrophoresis at room temperature.

After the electrophoresis, fluorescence detection (excitation wavelength635 nm, detection filter 650LP) was performed using a laser imagingscanner (Amersham Biosciences Corp., STORM 860). The results obtainedwere shown in FIG. 9.

As is clear from the results shown in FIG. 9, as the result ofperforming separation by polyacrylamide gel electrophoresis anddetection of the SLH reaction products obtained by the present inventionusing the above-described cy5delTINGC probe, it was shown that thedetection to distinguish each sample DNA which are different by fivenucleotides in the chain lengths of poly-T sequence could be performed.In addition, from these experimental results, showing highdiscriminatory performance between each sample, it could be anticipatedthat even if the difference by two nucleotides or even one nucleotide inpoly-T sequence length, could well be detected. Here, taking clinicalsignificance of diagnostic technique concerning TOMM40 gene polymorphisminto consideration, the population with poly-T sequence chain length inthe range of 11 to 16 nucleotides is classified as low risk group ofonset of Alzheimer's disease, and the population in the range of 19 to39 nucleotides, is discriminatory classified as a high-risk groups (ThePharmacogenomics Journal (2010) 10, 375-384). Therefore, the resultshown in this Example has suggested that such performance can exceed thestandard required for the diagnostic.

In addition, since polymorphism analysis of mono-, and di- ortri-nucleotide repeat have been difficult to be dealt with byconventional direct-sequencing method which is a in many cases, thesimple and highly precise measurement technology by the presentinvention is highly useful.

SEQUENCE LISTING

A computer-readable form (CRF) copy of the Sequence Listing for thisapplication has been submitted in a file, incorporated by referenceherein in its entirety, named SEQ_LISTING_15-792126_ST25.txt, which hasa creation date of Nov. 30, 2017, and a size of 8 kilobytes.

1-22. (canceled)
 23. A method for detecting whether a wild-type nucleotide region is deleted by a deletion mutation at a suspected mutated nucleotide region in a target DNA sequence in a sample, the method comprising: (1) providing a probe having a structure comprising 3′ 1stSS₂₁₁-X′₂₁₁-DM′-X′₂₁₂-2ndSS₂₁₁ 5′; wherein: the length of the probe is 30 to 300 nucleotides; 1stSS₂₁₁ is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 5′ side of the suspected deletion mutation region and 2ndSS₂₁₁ is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 3′ side of the suspected deletion mutation region; each of X′₂₁₁ and X′₂₁₂ independently represents a deoxyribonucleotide A, T, C or G; and DM′ is a nucleotide sequence 4 to 200 nucleotides in length that hybridizes with the region of the target DNA sequence that is present in the target DNA with a wild-type sequence and deleted from the target DNA with the deletion mutation, DM′ has the same length as the length of the suspected deletion mutation region, and DM′ contains a nucleotide sequence of 4 to 60 nucleotides that is capable of forming a double-stranded stem structure of 2 to 20 base pairs when the probe is hybridized with the target DNA sequence; (2) contacting the probe with the sample containing the target DNA sequence in a solution under conditions capable of forming probe/target DNA sequence hybrids, wherein there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation; (3) analyzing the probe/target DNA sequence hybrids formed in step (2) by an electrophoresis which is performed under conditions in which there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation, thereby obtaining an electrophoresis analysis result; and (4) determining whether the wild-type nucleotide region is deleted by the deletion mutation at the suspected mutated nucleotide region in the target DNA sequence in the sample by comparing the electrophoresis analysis result of step (3) with a first electrophoresis analysis result when the target DNA sequence has the wild-type sequence or/and a second electrophoresis analysis result when the target DNA sequence has the deletion mutation, wherein the first electrophoresis analysis result or/and the second electrophoresis analysis result is/are obtained by performing steps (1), (2) and (3).
 24. The method according to claim 23, wherein the electrophoresis is microchip electrophoresis or capillary gel electrophoresis.
 25. A method for detecting whether a wild-type nucleotide region is deleted by a deletion mutation at a suspected mutated nucleotide region in a target DNA sequence in a sample, the method comprising: (1) providing a probe having a structure comprising 3′ 1stSS₂₂₁-X′₂₂₁-DM′-Stp₂₂₁-X′₂₂₂-2ndSS₂₂₁ 5′; wherein: the length of the probe is 30 to 300 nucleotides; 1stSS₂₂₁ is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 5′ side of the suspected deletion mutation region and 2ndSS₂₂₁ is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 3′ side of the suspected deletion mutation region; each of X′₂₂₁ and X′₂₂₂ independently represents a deoxyribonucleotide A, T, C or G; DM′ is a nucleotide sequence 1 to 200 nucleotides in length that hybridizes with the region of the target DNA sequence that is present in the target DNA with a wild-type sequence and deleted from the target DNA with the deletion mutation, DM′ has the same length as the length of the suspected deletion mutation region, and DM′-Stp₂₂₁ contains a nucleotide sequence of 4 to 60 nucleotides that is capable of forming a double-stranded stem structure of 2 to 20 base pairs when the probe is hybridized with the target DNA sequence; (2) contacting the probe with the sample containing the target DNA sequence in a solution under conditions capable of forming probe/target DNA sequence hybrids, wherein there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation; (3) analyzing the probe/target DNA sequence hybrids formed in step (2) by an electrophoresis which is performed under conditions in which there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation, thereby obtaining an electrophoresis analysis result; and (4) determining whether the wild-type nucleotide region is deleted by the deletion mutation at the suspected mutated nucleotide region in the target DNA sequence in the sample by comparing the electrophoresis analysis result of step (3) with a first electrophoresis analysis result when the target DNA sequence has the wild-type sequence or/and a second electrophoresis analysis result when the target DNA sequence has the deletion mutation, wherein the first electrophoresis analysis result or/and the second electrophoresis analysis result is/are obtained by performing steps (1), (2) and (3).
 26. The method according to claim 25, wherein the electrophoresis is microchip electrophoresis or capillary gel electrophoresis. 